The AKAV isolate AKAV-TJ2016 (GenBank accession numbers: MT761689.1, MT761688.1, MT755621.1), BHK-21, Vero, and BSR-T7/5 cells were all stored in our laboratory.

Alexa Fluor 568- and 488-conjugated antibodies (A-11001、A-11011), DMEM (12100046), and Lipofectamine™ 3000 transfection reagent (L3000075) were purchased from Thermo Fisher Scientific. HRP-labeled goat anti-rabbit IgG (ZB-2301), goat anti-mouse IgG (ZB-2305), and β-actin antibody (TA-09) were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China). The Nano-Glo^® HiBiT Lytic Detection System and Nano-Glo^® HiBiT Blotting System kits (N3030、N2410) were purchased from Promega. The ClonExpress^® II One Step Cloning Kit (C112-01) were purchased from Vazyme Biotech Co., Ltd. The FastKing cDNA First Strand Synthesis Kit (KR116-01) was purchased from Tiangen Biotech (Beijing, China). Favipiravir (T-705, HY-14768), Ribavirin (ICN-1229, HY-B0434), and Remdesivir (GS-5734, HY-104077) were purchased from MedChemExpress LLC.

Overlapping PCR was used to clone the HiBiT gene to the C terminus of L, M, and S segments. All the primers were listed in Table 1. BHK-21 cells were transfected with the plasmids using Lipofectamine™ 3000 according to manufacturer’s instructions. Specifically, two sterile 1.5 mL centrifuge tubes were prepared, each containing 125 μL Opti-MEM. To one tube, 5 μL Lipofectamine 3000 was added, while the other tube received 2.5 μg plasmid DNA and 5 μL P3000™ Enhancer. Both tubes were thoroughly mixed, combined, and incubated at RT for 15 min. The resulting mixture was added dropwise to the pre-seeded cell culture plates. After 12 h, the medium was replaced with DMEM supplemented with 2% FBS, and the cells were incubated for an additional 72 h.

BHK-21 cells were fixed with 200 μL pre-cooled absolute ethanol at RT for 30 min followed by three washes with PBS (5 min each). Primary antibody was added and incubated at 37 °C for 60 min; after three PBS washes, secondary antibody was added and incubated at 37 °C for 60 min followed three PBS washes. The cells were observed under an inverted fluorescence microscope.

Viral stocks or AKAV-infected cells were collected, aliquoted, and added to 96-well plates (50 μL/well). The Nano-Glo^® HiBiT Lytic Reagent was prepared according to the manufacturer’s instructions and equilibrated to RT. Then, 50 μL reagent was added to each well containing the virus. The plate was shaken (300–600 rpm) for 10 minutes. After incubation, luminescence was measured using a multifunctional plate reader equipped with a luminescence module.

BHK-21 cells in 24-well plates were infected with rAKAV-L-HiBiT at 0.1 or 5 MOI. The whole samples were collected at 12, 24, 36, 48, 60, and 72 hpi, respectively. After repeated freeze-thaw cycles, the supernatants were harvested and the virus titers were measured by TCID₅₀ according to the Reed-Muench method. The titer of each sample was determined at least three replicates. Meanwhile, 50 μL of each viral samples were subjected to luciferase activity assay.

BHK-21 cells were washed twice with PBS and lysed in cell lysis buffer containing protease and phosphatase inhibitors. The lysates were centrifuged, and the supernatants were mixed with loading buffer and denatured in boiled water. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 2% BSA and then incubated with primary antibody at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at RT for 1 h. The membranes were detected using ECL substrate, and band intensities were quantified using Image J software. HiBiT-tagged proteins on nitrocellulose membranes were detected via Western blot using the Nano-Glo^® HiBiT Blotting System. The membranes were incubated with TBST for 4 h, followed by Nano-Glo^® HiBiT Blotting Reagent and overnight incubation at 4 °C. After adding the substrate, membranes were incubated at RT for 5 min and imaged with a chemiluminescence imager.

For the AKAV neutralization assay, hyperimmune serum (prepared and stored in our lab) was first two-fold serially diluted in DMEM (3 replicates per dilution). Concurrently, 50 μL of AKAV stock (100 TCID₅₀/mL) was mixed with 50 μL of each diluted serum, and the mixtures were incubated for 1 h. Subsequently, each mixture was added to BHK-21 cells, followed by an additional 72 h in an incubator. Finally, the whole cells were subjected to IFA, and their luciferase activity was measured using a multifunctional microplate reader.

BHK-21 cells seeded in 48-well plates were inoculated with rAKAV-L-HiBiT at an MOI of 0.1. Following 1 h of adsorption, the inoculum was removed and replaced with DMEM with 2% FBS and containing Favipiravir, Remdesivir, or Ribavirin at concentrations of 10 μM, 50 μM, or 100 μM, respectively. After 24 h of culture, cells were subjected to IFA to monitor the propagation of AKAV. Meanwhile, the supernatants were subjected to luciferase activity assay.

Statistical analysis was performed using GraphPad Prism software (version 10.0). Statistical differences were assessed using one-way ANOVA, two-way ANOVA, or unpaired t-tests. Significance is indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

This work was supported by the National Key R&D Program of China (2022YFD1802000), Youth Program of Beijing Natural Science Foundation (6254044), and Youth Talent Cultivation Project of Beijing Association for Science and Technology at Beijing University of Agriculture (BUA-QNTJ-2024005).

The AKAV genome comprises L, M, and S segments, all of which have been cloned into the plasmid TVT7R, respectively (Chen et al., 2021). We successively inserted the 33-nucleotide HiBiT at the C-terminus of the L, Gc, and N genes to generate three recombinant plasmids: TVT7R-L-HiBiT, TVT7R-M-HiBiT, and TVT7R-S-HiBiT (Figure 1A). Then, the three plasmids, only one of which carries the HiBiT tag gene, were co-transfected into BSR-T7/5 cells based on the three-plasmid infectious clone system (Chen et al., 2021). At 36 hours post-transfection, the cells were harvested and passaged three times. CPE and the N signals were observed only in cells co-transfected with the TVT7R-L-HiBiT, TVT7R-M, and TVT7R-S plasmids (Figure 1B). Then, the presence of HiBiT tags was confirmed by RT-PCR and subsequent Sanger sequencing (Figures 1C, D). Additionally, IFA was performed using an anti-N monoclonal antibody to monitor the replication efficiency of the virus in Vero cells over time (Figure 1E). The results indicated that rAKAV-L-HiBiT was successfully generated and could replicate efficiently in vitro.

To evaluate the stability of the HiBiT sequence, rAKAV-L-HiBiT was serially passaged 10 times in BHK-21 cells. RT-PCR analysis of the recombinant virus from passages 1 to 10 confirmed the presence of HiBiT in the viral genome (Figure 2A), and Sanger sequencing of the 10th passage further verified the integrity of HiBiT (Figure 2B). Next, the expression of the L-HiBiT recombinant protein was detected by Western blot and luciferase activity assay. As shown in Figure 2C, the L-HiBiT recombinant protein was highly expressed at 24 hpi. Additionally, the luciferase activity was approximately 2213-fold higher than that of cells infected with wild-type (WT) AKAV at 24 hpi (Figure 2D).

To assess the replication capacity of rAKAV-L-HiBiT, its one-step and multi-step growth curves were determined. As shown in Figures 2E, F, viral titers of rAKAV-L-HiBiT were comparable to those of WT AKAV. Collectively, these results indicate that the HiBiT fused to the C-terminus of the L gene is stable and does not impair viral replication. Then, we analyzed the correlations between rAKAV-L-HiBiT titers and HiBiT luciferase activity. First, the viral stocks were serially diluted 10-fold (from 1 to 10^-6), and the luciferase activity of diluted viral stocks was analyzed. The results demonstrated a strong correlation between luciferase activity and viral titers, with a correlation coefficient (r) of 0.9921(Figure 2G). To monitor luciferase activity during infection, BHK-21 cells were inoculated with rAKAV-L-HiBiT, and both the viral titers and luciferase activity were detected at 12, 24, 36, 48, and 60 hpi. As shown in Figure 2H, viral titers reached a plateau at 36 hpi and exhibited a positive correlation with luciferase activity. Notably, the luciferase activity increased gradually from 48 to 60 hpi, whereas the viral titers remained unchanged. This discrepancy may result from the accumulation of L-HiBiT fusion proteins in non-infectious viral particles or intracellular L-HiBiT protein.

While IFA remains the gold standard for quantifying neutralizing antibodies, its labor-intensive nature limits its utility. To establish a rapid alternative method, we evaluated the utility of rAKAV-L-HiBiT in neutralization assays. First, rAKAV-L-HiBiT stocks were titrated using both IFA and luciferase-based assay (LBA). As shown in Figures 3A, B, the viral titers showed no significant difference between the two methods.

To assess the suitability of rAKAV-L-HiBiT as a model virus, hyperimmune serum was used to neutralize both the WT AKAV and rAKAV-L-HiBiT. As shown by IFA and luciferase activity, viral replication was significantly inhibited in cells treated with the hyperimmune serum, indicating that the serum is appropriate for neutralization assays (Figures 3C, D). Subsequently, both rAKAV-L-HiBiT and WT AKAV were tested in hyperimmune serum neutralization assays. The results revealed no difference in neutralization rates between the two viruses, confirming that rAKAV-L-HiBiT can effectively serve as a model virus (Figure 3E).

To evaluate the suitability of rAKAV-L-HiBiT for antiviral drug screening, three antiviral compounds—ribavirin, remdesivir, and favipiravir —were tested in this study. In AKAV-infected groups, cells exhibited typical CPE at 36 hpi. In contrast, CPE was less severe in groups treated with ribavirin or remdesivir (Figures 4A, B). Notably, both remdesivir and ribavirin almost completely inhibited viral replication at a concentration of 150 μM, whereas favipiravir provided relatively weaker protection (Figure 4C).

We then detected luciferase activity of rAKAV-L-HiBiT in samples treated with the drugs. As shown in Figure 4D, 10 μM ribavirin reduced luciferase intensity by over 4-fold at 24 hpi, while increasing the concentration to 100 μM resulted in a more significant >30-fold reduction. Remdesivir inhibits virus replication similarly to ribavirin (Figure 4E), while favipiravir-treated cells showed less reduction in luciferase activity than the other two drugs (Figure 4F). This result provides a reliable quantitative readout but also confirms that the luciferase-tagged recombinant virus serves as a robust tool for high-throughput screening of anti-AKAV drugs.