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. 2024 Mar 16;39(3):422–433. doi: 10.1016/j.virs.2024.03.004

Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research

Rui Yu a,1, Xiaohong Li a,1, Peng Zhang c,1, Minghao Xu d, Jitong Zhao d, Jingjing Yan g, Chenli Qiu b,e, Jiayi Shu f, Shuo Zhang b, Miaomiao Kang b, Xiaoyan Zhang b,⁎, Jianqing Xu b,⁎, Shuye Zhang a,b,⁎
PMCID: PMC11279724  PMID: 38499155

Abstract

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.

Keywords: Enterovirus, Infectious clones, Reporter genes, Luciferase, Drug screening

Highlights

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    The incorporation of HiBiT-tag into enteroviruses greatly enhances the efficiency of rescuing recombinant viruses.

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    The virological characteristics of HiBiT-tagged enteroviruses are similar to those of wild type enterovirus viruses.

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    The HiBiT-tagged enteroviruses exhibit remarkable genetic stability.

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    The HiBiT-tagged enteroviruses facilitate the screening of antiviral drugs without affecting the sensitivity.

1. Introduction

Enterovirus is a positive-strand RNA virus belonging to the Picornavirus family (Jubelt and Lipton, 2014). The genus Enterovirus comprises 13 species, of which seven are known to infect humans, including Enterovirus A–Enterovirus D and Rhinovirus A–Rhinovirus C (Simmonds et al., 2020), collectively encompassing near 300 types (Sinclair and Omar, 2023). The viral genome is approximately 7.5 ​kb in length and consists of a 5′-untranslated region (5′ UTR), an open reading frame (ORF), and a 3′-untranslated region (3′ UTR). The ORF encodes a polyprotein that undergoes cleavage to form four structural proteins (VP4, VP3, VP2, and VP1) and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) (Chua et al., 2022). The second amino acid (glycine) of VP4 is linked to myristic acid, and myristoylated glycine is pivotal to enterovirus infection and capsid-membrane structure interaction (Cao et al., 2020). Enterovirus-associated hand-foot-mouth disease (HFMD), is a common illness caused by enterovirus infection, primarily affecting children under 5 years old (Chiu et al., 2016). Enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) are the main culprits behind acute HFMD cases (Kimmis et al., 2018), although coxsackievirus A4–7 (CVA4–7), coxsackievirus A9 (CVA9), coxsackievirus A10 (CVA10), coxsackievirus B1-3 (CVB1-3), and coxsackievirus B5 (CVB5) can also cause HFMD (Sarma, 2013). Inactivated vaccines against EV-A71 have been successfully developed by Beijing Vigoo Co., Ltd., Sinovac Biotechnology Co., Ltd., and the Kunming Institute of the Chinese Academy of Medical Sciences, with the C4 genotype being utilized (Zhu et al., 2013; Zhou et al., 2016). However, studies have demonstrated that these EV-A71 inactivated vaccines lack cross-reactivity against other HFMD pathogens (Chou et al., 2013). Therefore, there is an urgent need to further investigate the characteristics of different enteroviruses and explore the development of broad-spectrum antiviral agents.

Reporter genes in viruses can be valuable tools for studying viral pathogenesis, screening antiviral drugs, and evaluating neutralizing antibodies. However, constructing multiple reporter viruses covering most enteroviruses is challenging due to the small size of the enterovirus genome (about 7.5 ​kb). For instance, while EV-A71 (Shang et al., 2013) and CVA16 (Deng et al., 2015) carrying the eGFP reporter gene have been reported, CVA10-eGFP and CVA7-eGFP have not been documented. Similarly, although EV-A71-Gluc (Xu et al., 2015) using Gaussia luciferase (Gluc) has been constructed, CVA16-Gluc and CVA10-Gluc have not been reported. Another challenge is the genetic stability of reporter viruses. Numerous studies have focused on the stability of foreign genes in reporter viruses. For example, DsRed-EV-A71 and eGFP-EV-A71 were lost after six passages (Shang et al., 2013) and eGFP-Zika and NLuc-Zika exhibited changes in the phenotype of infected foci 96 ​h post-infection (Yun et al., 2020), indicating their instability.

Compared to other luciferase reporter genes, NanoLuc luciferase (NLuc) is significantly smaller and offers numerous advantages. In our previous study, we successfully constructed a CVA16-NLuc recombinant virus, demonstrating the applicability of the NLuc reporter gene to Coxsackievirus A16 (CVA16) (Yu et al., 2022). However, it remains to be determined whether the NLuc reporter gene can be applied to other viruses belonging to the species Enterovirus A (EVAs). In addition, the HiBiT peptide tag, consisting of 11 amino acids, has a strong affinity for a larger subunit called LgBiT, resulting in the formation of NLuc and subsequent generation of bright luminescence (Gaspar et al., 2020). Several research teams have successfully developed viruses using the HiBiT tag. For example, HiBiT-tagged infectious bronchitis virus (IBV-HiBiT) was created and remained genetically stable for over 20 passages in cell culture (Liang et al., 2020). Another successful application is the rescue of recombinant HiBiT-HBV, which enables the complete viral life cycle and facilitates high-throughput screening for HBV infection in vitro using supernatants (Sumiyadorj et al., 2022). Recently, the HiBiT-tagged reporter H3N2 influenza A virus (Zhang et al., 2023) and hepatitis E virus (Nagashima et al., 2023) have also been constructed and have been successfully utilized for the screening and evaluation of antiviral drugs. Additionally, the HiBiT tag has been utilized in the screening of neutralizing antibodies against SARS-CoV-2 (Miyakawa et al., 2020). These studies collectively demonstrate the suitability of the HiBiT tag, given its small size, for constructing infectious clones. However, there is currently a lack of reports regarding HiBiT-tagged enteroviruses.

In this report, we first verified the feasibility and versatility of the EVAs-NLuc infectious clones. However, our initial attempts to introduce a NLuc reporter gene into EV-A71, CVA10, and CVA7 failed to rescue viable progenies. Subsequently, we successfully constructed four HiBiT-tagged infectious clones of EV-A71, CVA16, CVA10, and CVA7 and rescued EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT viruses. We then characterized the EVAs-HiBiT along with their wild-type enteroviruses (EVAs-WT), and our results showed that all the recombinant EVAs-HiBiT exhibited comparable growth characteristics to the wild-type viruses in vitro. Furthermore, we compared the virological differences between CVA16-NLuc and CVA16-HiBiT recombinant viruses. Notably, the CVA16-HiBiT recombinant virus displayed higher infectivity and replication speed compared to the CVA16-NLuc recombinant virus. Additionally, our data indicated that the HiBiT tag in all four EVAs-HiBiT maintained stable expression over ten passages in cultured cells without reducing the luciferase activity. Finally, we tested the application of EVAs-HiBiT in the evaluation of antiviral activity of drugs. We measured the IC50 of dibucaine, micafungin, suramin, and itraconazole for EV-A71, CVA16, CVA10, and CVA7, demonstrating their promising potential for high-throughput screening of EVAs-HiBiT.

2. Material and methods

2.1. Cells and virus

The African green monkey kidney (Vero) cells, human hepatocellular carcinoma (Huh-7) cells, human malignant embryonic rhabdomyoma cells (RD), and Henrietta Lacks strain of cancer cells (HeLa) used in this study were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 ​U/mL of penicillin, and 100 ​μg/mL of streptomycin at 37 ​°C in the presence of 5% CO2.

EV-A71 (Genbank: HQ882182) and CVA16 (Genbank: OP293089) are clinical isolates, while CVA10 (Kowalik strain, GenBank: AY421767.1) and CVA7 (Parker strain, GenBank: AY421765.1) are prototype strains that were rescued by reverse genetics. The EV-A71, CVA16, CVA10, and CVA7 viruses were cultured in DMEM supplemented with 2% FBS, 100 ​U/mL of penicillin, and 100 ​μg/mL of streptomycin at 37 ​°C in the presence of 5% CO2. The CVA16 strain OP293089 was isolated in the Biosafety Laboratory of Shanghai Public Health Clinical Center, and the EV-A71 strain HQ882182.1 was obtained from the Fujian Center for Disease Control. The CVA10 strain AY421767.1 and CVA7 strain AY421765.1 were prototypes, and these two viruses were rescued by reverse genetics in our laboratory.

2.2. Wide-type enterovirus infectious clones

The construction of CVA10 and CVA16 infectious clones has been reported previously (Wang et al., 2020; Yu et al., 2022). The construction strategy for CVA7 and EV-A71 infectious clones was similar. Specifically, the EV-A71 genome RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, France), and reverse transcription (Promega, France) was performed using random primers to obtain viral genome cDNA. Then, three DNA fragments (F1, F2 and F3) covering the complete EV-A71 genome were amplified by PCR using virus-specific primers with the cDNA as the template. The F2 fragment contained homologous arms related to the F1 and F3 sequences, while the F1 fragment contained homologous arms related to the vector sequence. As for the prototype CVA7, three DNA fragments covering the CVA7 virus genome were chemically synthesized (GenBank: AY421765.1), and each fragment also contained approximately 20 bp homologous arms for seamless cloning. In addition, the vector fragment was obtained by PCR using primers with homologous arms at the 5′ end, using the CVA16 infectious clone as the template. The vector DNA fragment contained the T7 promoter and Hhrib sequence at the 3′ end, and the poly(A) sequence and a sequence homologous to the end of the EV-A71 or CVA7 virus genome at the 5′ end for homologous recombination with the virus genome. Subsequently, the virus fragments were recombined with the vector fragments using the ClonExpress II One Step Cloning Kit (Vazyme, China), sequentially assembled, and loaded onto the vector. The recombinant products were transformed into Trans2-Blue competent cells (TransGen, China), single clones were picked, and after sequencing verification, they were stored at −20 ​°C for future use. The primers involved in the construction of infectious clones described above are listed in Supplementary Table S1.

2.3. Construction of NanoLuc (NLuc) or HiBiT-tagged infectious clones

The construction of the reference infectious clones with an NLuc reporter gene was based on our previous research (Yu et al., 2022). To insert the HiBiT sequence between the 5′ UTR and VP4 of the wild-type EV-A71, CVA16, CVA10, and CVA7 infectious clones, three additional amino acids with synonymous mutations and a 2A protease recognition site (AITTL) were simultaneously inserted. The Tag, AITTL, and three additional amino acid sequences were introduced using PCR primers (GENEWIZ, China) in a seamless cloning method. Specifically, the wild-type infectious clones were used as DNA templates for PCR amplification using 2 ​× ​Hieff Canace® Gold PCR Master Mix (Yeasen, China). The amplified products were purified from agarose gel and transferred into Trans2-Blue competent cells (TransGen, China) for activation and overnight culture. Single colonies were selected for sequencing verification, and correctly sequenced plasmids were stored at −20 ​°C. The detailed primers for clone construction can be found in Supplementary Tables S2 and S3.

2.4. Virus rescue

To generate the EVAs-NLuc and EVAs-HiBiT viruses from the EVAs-tagged infectious clones, we utilized the T7 transcription kit (Novoprotein, China) for in vitro transcription following the manufacturer's instructions. The resulting mRNA was then purified using a LiCl solution. For the transfection, 2 ​μg of purified viral mRNA was introduced into Vero cells in 6-well plates using Lipo3000 (Thermo Fisher Scientific, US). After a 6 h incubation at 37 ​°C, the medium was replaced with fresh medium containing 2% FBS. Once a significant cytopathic effect was observed, the cell culture supernatant was collected by centrifugation at 1000 ×g for 5 ​min after two freeze–thaw cycles. If no obvious cytopathic effect was observed, the cell culture supernatant was still collected using the same method for subsequent virus passage and further verification.

2.5. Plaque assay

The titer of virus was determined by plaque assay as previously described (Chen et al., 2023). Twelve ​hours before the experiment, 1.5 ​× ​105 Vero cells were seeded onto 12-well plates. The EVAs-HiBiT and EVAs-NLuc and their respective wild-type viruses were serially diluted 10-fold and added to the cells. The plates were gently shaken every 15 ​min and incubated at 37 ​°C for 2 ​h. After incubation, the cells were washed twice with PBS and then loaded with 2 ​× ​DMEM (4% FBS) mixed with an equal volume of 2.4% Avicel (IMCD, China). The plates were further incubated at 37 ​°C for an additional 2–3 days. To visualize the infected cells, the plates were fixed with 4% paraformaldehyde for 2 ​h and stained with 1% crystal violet for another 1 ​h. Subsequently, the plates were rinsed with running water and photographs were taken for further analysis.

2.6. Luciferase activity assay

The replication of EVAs-HiBiT virus and EVAs-NLuc virus in cells can be characterized by the expression levels of the inserted NLuc and HiBiT. Specifically, to detect the replication of the virus in infected cells, remove the cell supernatant and add 50 ​μL of PBS (for a 96-well plate). Then, add the prepared luminescent substrate. For the detection of EVAs-NLuc virus, add 50 ​μL of Nano-Glo HiBiT lytic detection solution (Promega, USA) and for EVAs-HiBiT, add 50 ​μL of Nano-Glo® HiBiT Lytic Reagent (Promega, USA). Shake thoroughly for 5 ​min under light-protected conditions, and then measure the luminescence intensity in a black bottom plate according to the instructions.

2.7. Flow cytometry

2.0 ​× ​105 RD or 1.0 ​× ​105 Vero cells were seeded on 24-well plates and incubated overnight. The cells were then infected with the HiBiT-tagged EVAs and wild-type viruses at a multiplicity of infection (MOI) of 1 for 6 ​h. After infection, the cells were harvested and fixed with 100 ​μL of Fixation/Permeabilization solution (BD Cytofix/Cytoperm Kit, USA) for 20 ​min on ice. Following fixation, the cells were washed twice with 500 ​μL of 1 ​× ​BD Perm/Wash™ buffer for 5 ​min each. Next, the cells were stained with 50 ​μL of dsRNA antibody (Cell Signaling Technology, USA) for 30 ​min in the dark, followed by two washes with 500 ​μL of 1 ​× ​BD Perm/Wash™ buffer for 5 ​min each. Finally, the cells were stained with 50 ​μL of donkey anti-mouse IgG (H ​+ ​L) secondary antibody (Invitrogen, USA) for 30 ​min in the dark and kept on ice. The fluorescence signal from single cells was detected using a BD LSRFortessa™ instrument, and the data were analyzed using Flowjo software.

2.8. Extraction and quantification of intracellular viral RNA

After viral incubation, the cell culture medium was discarded, and the cells were washed three times with PBS. RNA extraction and purification of intracellular viral RNA were performed by adding RNAzol (MRC, USA) to lyse the cells, followed by extraction using the Direct-zol™ RNA MiniPrep w/Zymo-Spin™ IIC Columns (Zymo, USA). Total RNA was obtained and used as a template for quantitative reverse transcription PCR (RT-qPCR) using the HiScript II One Step RT-qPCR SYBR Green Kit (Vazyme, China) with virus-specific qPCR primers. The viral RNA was quantified based on a standard curve, allowing for the calculation of viral copy numbers. All qPCR primers involved in this study have been listed in Supplementary Table S4.

2.9. Virus growth competition experiment

Vero cells (1 ​× ​105) were seeded in a 24-well plate and cultured overnight to reach approximately 80% confluency. An equal amount of CVA16-HiBiT and CVA16-NLuc viruses were thoroughly mixed and used to infect the cells for 1 ​h. Afterward, the cells were washed three times with PBS, fresh culture medium was replaced, and they were placed in a 37 ​°C incubator. Virus RNA was extracted at 2 ​h and 6 ​h post-infection using the aforementioned method, with GAPDH serving as an internal reference gene. The relative quantification of the RNA from both viruses was performed using virus-specific qPCR primers listed in Supplementary Table S4.

2.10. Genetic stability experiment

RD, HeLa, Huh-7, and Vero cells were seeded in 12-well plates at a density of 3 ​× ​105 ​cells per well. After 12 ​h of incubation, the cell culture supernatant was discarded and the cells were washed once with PBS. Then, 500 ​μL of DMEM containing 2% FBS and 500 ​μL of P0 generation EVAs-HiBiT viruses were added to the cells, followed by incubation at 37 ​°C with 5% CO2. After 2 days, the supernatant was harvested by freeze-thawing twice and centrifuging at 2000 ×g for 5 ​min. Subsequently, 500 ​μL of DMEM containing 2% FBS and 500 ​μL of P1 EVAs-HiBiT cell culture lysates were used to infect the cells. This procedure was repeated until 10 generations of EVAs-HiBiT virus were harvested. Finally, 20 ​μL of P2, P4, P6, P8, and P10 viruses were used to infect RD, HeLa, Huh-7, and Vero cells, respectively, in 96-well plates, and luciferase activity was detected.

2.11. Antiviral assay

To determine the sensitivity of EVAs-HiBiT viruses to four drugs, 2.5 ​× ​104 RD were seeded in 96-well black plates and incubated overnight. For virus post-entry inhibitors such as Micafungin, Dibucaine, and Itraconazole, different concentrations of drugs were prepared in 2% FBS DMEM and incubated with cells at 37 ​°C for 1 ​h before infecting with the virus at an MOI of 0.01. For virus binding inhibitors like Suramin, the virus was first incubated with different concentrations of drugs at 37 ​°C for 1 ​h before adding the virus-drug mixture to the pre-seeded cells. After 24 ​h of infection, luciferase activity was measured to determine the inhibition rate.

For wild-type enteroviruses, we used flow cytometry to determine the antiviral activity of drugs. Specifically, 2.0 ​× ​105 RD cells were seeded in 24-well plates and incubated overnight. The virus infection was carried out following the same infection protocol as described above, consistent with the mechanism of action of the drugs. After 24 ​h, infected cells were collected, and intracellular viral dsRNA was stained, and viral infection rate was analyzed by flow cytometry.

2.12. Cell viability analysis

2.5 ​× ​104 RD and Vero cells were seeded in 96-well plates and incubated overnight. After treatment with different concentrations of chemical inhibitors, cell viability was assessed using the Cell Counting Kit-8 (CCK-8 Kit, Yeasen, China). Specifically, dibucaine, micafungin, suramin, and itraconazole were diluted with 2% FBS DMEM and added to triplicate wells at each concentration. The plates were then incubated at 37 ​°C for 24 ​h. After discarding the cell supernatant, 100 ​μL of a 10-fold diluted CCK-8 solution in DMEM was added to each well. The plates were incubated at 37 ​°C for 1 ​h, and the absorbance at 450 ​nm was measured using the Cytation5 microplate reader (Biotek, USA).

2.13. Statistical analysis

The data were analyzed using Prism 7.0 software (GraphPad, USA) and presented as means ​± ​SEM. Statistical significance between the two groups was determined using an unpaired two-tailed Student's t-test. Differences were considered not significant (ns) P ​> ​0.05; ∗, P ​< ​0.05; ∗∗, P ​< ​0.0001.

3. Results

3.1. Development and characteristic of EVAs-NLuc recombinant viruses

In our previous study, we successfully constructed a recombinant CVA16 virus carrying the NLuc reporter gene and demonstrated its utility in antiviral drug screening and neutralizing antibody detection (Yu et al., 2022). However, it remains to be determined whether the NLuc reporter gene can be applied to other viruses of the species Enterovirus A. Therefore, we attempted to insert the NLuc gene into other enterovirus genomes to generate recombinant enteroviruses. We successfully inserted the NLuc gene into EV-A71, CVA7, CVA10, and CVA16 infectious clones, which were verified by sequencing. Through in vitro transcription, we obtained viral genomic RNA and transfected it into Vero cells in order to obtain recombinant viruses carrying NLuc (Fig. 1A). We used CVA16-NLuc as a positive control to ensure the reliability of the experimental system. However, no apparent cytopathic effect (CPE) was observed after transfection of the other enterovirus-NLuc RNAs into cells, except for CVA16-NLuc (Fig. 1B). Based on our previous experience, we hypothesized that a small number of rescued recombinant viruses might be present and would require continuous passaging to restore their virulence. Therefore, we performed serial passaging of these recombinant viruses and monitored the viral titers and NLuc gene expression at different generations. To determine the presence of recombinant viruses, we performed plaque assays and tested NLuc reporter gene expression every two generations. Unfortunately, according to the results of the plaque assays, no plaques were observed from P2 to P10 for EV-A71-NLuc, CVA7-NLuc, and CVA10-NLuc, while visible plaques were observed for the positive control CVA16-NLuc (Fig. 1C). Moreover, when we infected cells with virus supernatants collected from different generations and detected NLuc gene expression within the cells, similar results were obtained: except for cells infected with CVA16-NLuc, no positive signal was detected within the cells infected with other recombinant viruses (Fig. 1D). In summary, our study data clearly indicate that, except for CVA16, insertion of NLuc into the other tested enteroviruses resulted in the failure to rescue these recombinant viruses.

Fig. 1.

Fig. 1

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Construction of EVAs-NLuc infectious clones and rescue of recombinant virus. A Schematic representation of the construction of EVAs-NLuc infectious clones and the strategy to rescue recombinant EVAs-NLuc. Viral genomic fragments were obtained by extracting the virus genome (if available) and reverse transcription (RT)-PCR or DNA synthesis. These fragments were then sequentially linked and cloned into the PL451 vector using seamless cloning to construct the wild-type infectious clone of enteroviruses. Subsequently, the NLuc gene and a 2A protease cleavage site located downstream were introduced by seamless cloning to obtain the EVAs-NLuc infectious clones. The linearized clones were used as templates for in vitro transcription to obtain EVAs-NLuc genomic RNA, which was then transfected into Vero cells. After 24–48 ​h of transfection, recombinant viruses were harvested by repeated freeze–thaw cycles and centrifugation, and they were continuously passaged to collect each generation of recombinant viruses for virological analysis. B The EVAs-NLuc RNA obtained from in vitro transcription was transfected into Vero cells in a 24-well plate, and the cytopathic effect (CPE) was observed after 24 ​h. Scale bar: 100 ​μm. C Viral plaque assay was utilized to determine the titers of P2, P4, P6, P8, and P10 EVAs-NLuc virus, with CVA16-NLuc showing plaque formation on Vero cells with a 104-fold dilution, and other EVAs-NLuc viruses showing no plaque formation even with no dilution of the viral stock. D 50 ​μL of P2, P4, P6, P8, and P10 EVAs-NLuc virus were inoculated onto Vero cells in a 96-well plate, and the intracellular NLuc activity was measured after 24 ​h.

3.2. Construction and characteristics of EVAs-HiBiT

We speculated that the failure to rescue the EVAs-NLuc may be attributed to the large size of the NLuc, which may have affected viral genome replication within cells. Therefore, selecting a smaller exogenous gene may be a feasible strategy. HiBiT, as a small subunit of NLuc consisting of only 11 amino acids, can function as a bioluminescent reporter in the presence of LgBiT. This small reporter gene may have less impact on virus replication and may be more suitable for constructing recombinant viruses.

To construct HiBiT-tagged infectious clones of EV-A71, CVA16, CVA10, and CVA7, the HiBiT tag was inserted after the first initiation codon in the P1 region. Additional three amino acids and a 2A protease recognition site (AITTL) were added to the N- and C- terminals of HiBiT respectively using a seamless cloning method (Fig. 2A). The PCR primers used for constructing HiBiT-tagged infectious clones are listed in Supplementary Table 3. EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT infectious clones were correctly sequenced. Through T7 in vitro transcription and mRNA purification, we transferred mRNA of EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT to cells and rescued four HiBiT-tagged enteroviruses. Both HiBiT-tagged viruses and wild-type viruses caused apparent cytopathic effects 24 ​h post infection (Fig. 2B). Additionally, similar plaque sizes and numbers were observed for the HiBiT-tagged viruses and wild-type viruses (Fig. 2C). Furthermore, we tested the performance of EVAs-HiBiT in single round infections and found that their infectivity was comparable to that of wild-type viruses (Fig. 2D), indicating similar infection rates. To further evaluate the differences in viral replication between the HiBiT-tagged viruses and wild-type viruses, we quantified the changes in viral copy numbers at different time points after infection using quantitative PCR. Our findings revealed that the growth rates of the tagged viruses were comparable to those of the untagged viruses (Fig. 2E–H). In addition, we also measured the expression of HiBiT in cells infected with EVAs-HiBiT using luciferase activity assays. The levels of HiBiT exhibited a similar time-dependent increase in cells infected with all four EVAs-HiBiT viruses, with the highest levels reached around 24 ​h (Fig. 2E–H), indicating that the expression of the exogenous gene is synchronized with the amplification of the viral genome. In summary, these results demonstrate that the insertion of the small HiBiT sequence has minimal impact on the infection and replication of enteroviruses.

Fig. 2.

Fig. 2

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Construction and characteristics of EVAs-HiBiT. A Schematic diagram of EVAs-HiBiT infectious clones. EVAs-HiBiT infectious clones were constructed by introducing the HiBiT sequence and a downstream 2A protease cleavage site into the wild-type EVAs infectious clones, as described in the Methods section. B Vero cells were infected with the wild-type EV-A71, CVA10, CVA7, CVA16, and EVAs-HiBiT viruses from the P1 generation, and cytopathic effect (CPE) was observed after 18 ​h. Scale bar: 100 ​μm. C Plaque formation of wild-type viruses and recombinant HiBiT-tagged viruses after infection of Vero cells. D RD cells were infected with the wild-type viruses and HiBiT-tagged viruses at an MOI of 1 for 6 ​h, and viral dsRNA was detected by flow cytometry. E–H RD cells were infected with EV-A71, CVA10, CVA7, CVA16, and their corresponding EVAs-HiBiT viruses at an MOI of 0.01. At 0, 6, 12, 18, 24, and 30 ​h, the viral genomic RNA load in the cells (left y-axis, solid line) and the expression level of HiBiT (right y-axis, dashed line) were measured. Data are means ​± ​SD of three replicate samples.

3.3. CVA16-HiBiT has a replication advantage over CVA16-NLuc

In our previous study, we reported the construction of a recombinant CVA16-NLuc virus carrying the NLuc gene (Yu et al., 2022). In this study, we inserted the HiBiT sequence at the same position in the viral genome and successfully rescued the CVA16-HiBiT recombinant virus. Here, we compared the virological differences between recombinant CVA16 viruses carrying different exogenous genes. We first evaluated the infectivity of CVA16-NLuc and CVA16-HiBiT in single-round infections. By staining the viral dsRNA, we found that CVA16-HiBiT exhibited significantly higher infectivity than CVA16-NLuc (Fig. 3A). This conclusion was further confirmed in plaque assays, as CVA16-HiBiT formed significantly larger plaques than CVA16-NLuc (Fig. 3B). To further demonstrate the differences in infection and replication between CVA16 recombinant viruses carrying two different reporters, we co-infected Vero cells with both viruses and quantified the viral genome levels using virus-specific qPCR primers. The results showed that at 2 ​h post-infection, the levels of CVA16-NLuc and CVA16-HiBiT genomes in cells were similar (20%–40% difference) under different MOI conditions (Fig. 3C). This result was consistent with our speculation, as the exogenous gene was inserted into the viral non-structural genes and would not be present on the viral capsid, thus not affecting the viral entry stage. However, at 6 ​h post-infection, the levels of CVA16-HiBiT genomes were significantly higher than those of CVA16-NLuc under all three MOI conditions (Fig. 3C). This further confirmed our hypothesis that the insertion of a larger exogenous gene may impose a burden on viral genome replication, mainly affecting the post-entry stage of the virus. Additionally, we monitored viral replication in cells infected with both recombinant viruses. The results showed that compared to CVA16-NLuc, CVA16-HiBiT exhibited faster replication rates and higher viral genome accumulation at 24 ​h post-infection (Fig. 3D). Similarly, the expression of the exogenous gene was correlated with viral genome replication, as luciferase activity in cells infected with CVA16-HiBiT was higher at different time points compared to CVA16-NLuc (Fig. 3E). Furthermore, we found that the titer of CVA16-HiBiT was approximately 20–40 times higher than that of CVA16-NLuc during viral amplification (data not shown). In conclusion, our study suggests that CVA16-HiBiT has a stronger replication capacity than CVA16-NLuc, which may be attributed to the impact of the larger exogenous gene insertion on viral genome replication. This further underscores the potential of “HiBiT” as an exogenous Tag in the development of reporter viruses.

Fig. 3.

Fig. 3

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Comparison of virological characteristics between CVA16-HiBiT and CVA16-NLuc. A Vero cells were infected with CVA16-HiBiT and CVA16-NLuc at an MOI of 1, and after 6 ​h of infection, viral dsRNA was examined by flow cytometry to determine the viral infectivity. B Plaque formation of CVA16-HiBiT and CVA16-NLuc after infection of Vero cells. C CVA16-HiBiT and CVA16-NLuc were mixed at MOI of 0.1, 0.5, and 1, and then used to infect Vero cells. After 2 and 6 ​h, cells were collected and RNA was extracted to quantify viral genomic levels relative to GAPDH as an internal reference, to calculate the relative expression levels of CVA16-HiBiT compared to CVA16-NLuc. D–E Vero cells were infected with CVA16-HiBiT and CVA16-NLuc at an MOI of 0.01, and at 0, 6, 12, 18, 24, and 30 ​h, the levels of viral genomic RNA in the cells (D) and the levels of intracellular NLuc or HiBiT activity (E) were measured. The t-test was used to compare differences. Data are means ​± ​SD of three replicate samples (∗P ​< ​0.05; ∗∗P ​< ​0.0001).

3.4. Validation of the stability of EVAs-HiBiT

Enteroviruses carrying a reporter gene often face challenges due to genetic instability, necessitating the investigation of the HiBiT tag's stability in the EVAs genome. We initially assessed the genetic stability of EVAs-HiBiT in two susceptible cell lines, RD and Vero. Serial passages of EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT were performed in RD and Vero cells. The luminescence of cell lysates from EVAs-HiBiT viruses at passages 2, 4, 6, 8, and 10 was measured after infecting RD and Vero cells in 96-well plates (Fig. 4A). The luminescence remained relatively stable with minor variations in infected cells of both cell lines. Replication of the virus in non-susceptible cells can be more challenging and may lead to the loss of exogenous insert sequences. To investigate this further, we evaluated the genetic stability of EVAs-HiBiT in two additional commonly utilized cell lines, HeLa and Huh-7. Encouragingly, we found that these four HiBiT-tagged enteroviruses displayed consistent genetic stability during replication in both cell lines (Fig. 4B). This indicates that the HiBiT tag in EVAs-HiBiT remained stable in different cell lines for up to 10 passages, demonstrating for the first time the stable culture of various recombinant enteroviruses using the same design principle in different cell lines. Furthermore, we also analyzed the stability of CVA16-NLuc in two susceptible cell lines, RD and Vero. We found that while CVA16-NLuc remained stable in Vero cells during passages, its expression gradually decreased in RD cells (Fig. 4C). Compared to passage 2, the luciferase activity of CVA16-NLuc decreased by more than two orders of magnitude in passage 10. These findings suggest that the stability of the exogenous gene NLuc in recombinant viruses is not as reliable as the smaller HiBiT tag, indicating that smaller exogenous genes may be more easily maintained and stable during the replication process in cells.

Fig. 4.

Fig. 4

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EVAs-HiBiT exhibits great genetic stability. A EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT from P2, P4, P6, P8, and P10 were used to infect RD and Vero cells. Luciferase activity was measured at 24 ​h after infection. B Passage stability of EVAs-NLuc in HeLa and Huh-7. C Passage stability of CVA16-NLuc in RD and Vero cells. Data are means ​± ​SD of four replicate samples.

3.5. Using EVAs-HiBiT in antiviral assays

EV-A71, CVA16, CVA10, and CVA7 are the main pathogens of HFMD, and there is an urgent need for broad-spectrum drugs for HFMD. To evaluate the suitability of EVAs-HiBiT as an antiviral assay, we selected four FDA-approved drugs (dibucaine, micafungin, suramin, and itraconazole) for the antiviral assay. Firstly, we assessed the toxicity of these drugs on RD and Vero cells using the CCK-8 assay. Dibucaine showed high cytotoxicity to RD and Vero cells at concentrations of 320 ​μM and 640 ​μM, respectively (Fig. 5A). Similarly, micafungin exhibited high toxicity to RD and Vero cells at concentrations of 128 ​μM and 256 ​μM, respectively (Fig. 5B). Surprisingly, even at a high concentration of 1280 ​μM, suramin did not significantly affect the cellular viability of RD and Vero cells (Fig. 5C). On the other hand, Itraconazole at a concentration of 1000 ​μM had no effect on Vero cells, while RD cells completely lost their cellular activity (Fig. 5D).

Fig. 5.

Fig. 5

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EVAs-HiBiT facilitates antiviral drug screening. A–D RD and Vero cells were treated with different concentrations of dibucaine, micafungin, suramin, and itraconazole in triplicate, and cell viability was assessed using the CCK-8 assay after 24 ​h. E–H Antiviral activity of dibucaine, micafungin, suramin, and itraconazole was tested using EVAs-HiBiT viruses. RD cells were seeded in black 96-well plates and incubated overnight. Relative light units (RLU) were measured 24 ​h after treatment with viruses (103 TCID50) and drugs, and inhibition rates were calculated. I Flow cytometry was performed to examine the inhibitory effects of micafungin and suramin on EV-A71 and CVA7, as described in the Methods section. The left panel shows representative graphs, and the right panel shows the statistical analysis. The infection rate was normalized and the control group was set as 100%. Data are means ​± ​SD of three replicate samples. J Four drugs were tested for their antiviral activity against EVAs-HiBiT in RD and Vero cells, and CC50, IC50, and SI values were calculated.

Next, we evaluated the antiviral activities of dibucaine, micafungin, suramin, and itraconazole using the EVAs-HiBiT system. The inhibition rates were calculated based on the decrease in luciferase activity. Dibucaine and itraconazole showed inhibitory effects on all four EVAs-HiBiT (Fig. 5E, H), while Micafungin and Suramin had little effect on CVA7 (Fig. 5F, G). To confirm that the insertion of HiBiT did not alter the sensitivity of CVA7 to dibucaine and itraconazole, we assessed the consistent effects of these drugs on wild-type CVA7, with EV-A71 used as a positive control. Our data revealed that compared to the control group, 30 ​μM Micafungin and 120 ​μM itraconazole completely inhibited the infection of EV-A71, and even lower concentrations of these drugs showed inhibitory effects on EV-A71 infection (Fig. 5I). However, the inhibitory effects of micafungin and itraconazole on CVA7 were limited. At concentrations of 30 ​μM Micafungin and 120 ​μM Itraconazole, the infection rate of CVA7 only decreased by around 20%, and lower concentrations of these drugs did not show any inhibitory effects on CVA7 (Fig. 5I). This result is consistent with the inhibitory effects of these two drugs on CVA7-HiBiT. Therefore, our results indicate that the insertion of HiBiT does not alter the sensitivity of CVA7 to these drugs. Furthermore, we calculated the IC50, CC50, and selection index (SI) of the four drugs and presented them in Fig. 5J. It is worth noting that some drugs did not show cytotoxicity even at the highest tested concentration, thus we could not calculate the CC50 and SI, which are represented by dashes in the table. Given that EVAs-HiBiT and wild-type enteroviruses exhibit consistent sensitivity to these drugs, EVAs-HiBiT can serve as a powerful tool for antiviral drug screening.

4. Discussion

Recombinant viruses carrying reporter genes have played a crucial role in simplifying virus detection in both basic and applied studies. When constructing recombinant reporter viruses for enteroviruses, two key factors must be considered: the insertion site and the choice of the reporter gene. The enteroviral genome can be roughly divided into three regions: the region responsible for structural protein expression, the non-structural protein region, and the untranslated regions (UTRs) located at both ends of the genome. Inserting the exogenous gene in the region responsible for structural protein expression may affect virus assembly and receptor binding, thus influencing virus yield and infectivity. On the other hand, inserting the exogenous gene in the non-structural protein region may impact non-structural protein expression or lead to protein functional defects, thereby affecting viral protein synthesis and genome replication. While the insertion of the exogenous gene in the UTRs does not directly affect the synthesis and function of viral proteins, it can still influence the conformation of adjacent translation and replication-related elements, potentially resulting in replication barriers of the viral genome. Previous studies have found that exogenous genes can be successfully inserted and expressed in various regions within the enteroviral genome, including the internal regions of structural protein genes (Arnold et al., 1996; Lyu et al., 2015), non-structural protein genes (Van Der Schaar et al., 2016; Wang et al., 2020), the region between structural and non-structural proteins (P1 and P2 regions) (Chu et al., 2013) as well as the junction between the 5′ UTR and the P1 (Shang et al., 2013; Deng et al., 2015; Xu et al., 2015). In terms of selecting a reporter gene, theoretically, the smaller the exogenous inserted gene, the less impact it has on the virus. Therefore, we focused our attention on HiBiT. It consists of only 11 amino acids and functions as a small subunit of the NanoLuc gene, allowing it to interact with LgBiT and exhibit luciferase activity. While HiBiT is usually used as a fusion tag with other proteins, inserting tags into the compacted enterovirus genome may disrupt viral functions and hinder virus rescues. Therefore, we hypothesized that the HiBiT tag could function as an independent unit. In this study, we inserted HiBiT after the 5′ UTR and before the P1 region, with a 2A protease cleavage site (AITTL) linking HiBiT and P1. In this design, HiBiT contains 11 amino acids, and with the addition of AITTL, a 16-amino acid peptide is formed in infected cells. Initially, it was uncertain whether HiBiT could be expressed properly and maintain the correct conformation for binding with LgBiT. However, the final results demonstrated the feasibility of this design.

Using HiBiT to construct infectious clones has several prominent advantages. Firstly, the success rate of rescuing HiBiT recombinant viruses is significantly improved. In this study, we attempted various constructions of EVAs-NLuc and found that even with the small NLuc, these attempts were mostly unsuccessful. However, when replaced with HiBiT, this situation was completely reversed, and the high success rate of rescuing EVAs-HiBiT recombinant viruses was unique. Additionally, while NLuc, GFP, DsRed, and Gluc can be used as exogenous inserted genes to construct recombinant enteroviruses, these reporter genes were often only compatible with a few viral strain. However, HiBiT recombinant viruses have a high success rate of rescue and strong compatibility, making it highly likely to be extended to other enteroviruses. Secondly, we compared EVAs-WT and EVAs-HiBiT through various methods, demonstrating that the insertion of HiBiT has minimal impact on the viral growth characteristics. Furthermore, we compared CVA16-NLuc and CVA16-HiBiT through virus growth competition experiments, revealing for the first time that the size of the exogenous gene significantly affects the replication of recombinant viruses in cells. Even with the use of the small-sized NLuc gene, it significantly inhibited the replication level of the virus. Thirdly, we discovered the extraordinary genetic stability of EVA-HiBiT. Genetic stability is one of the major issues limiting reporter gene viruses. Previous reports have shown that this is primarily due to gradual deletions occurring in the reporter gene during genome replication, resulting in loss of the exogenous gene (Shang et al., 2013), but high levels of luciferase activity could still be detected in HiBiT-tagged bronchitis coronavirus after transmission to P20 generation (Liang et al., 2020). As a result, when using reporter gene viruses, it is necessary to prepare primary passage viruses from infectious clones each time, rather than using recombinant viruses continuously passaged in cells. This makes the application process more cumbersome and increases the technical barrier. However, our EVAs-HiBiT exhibits high stability in various cell lines and can be continuously passaged in different cell lines without losing reporter activity. This lays the foundation for its widespread application.

Dibucaine, a commonly used local anesthetic in clinical practice (Shribman and Hanning, 1986), has been shown to inhibit CVB3, EVA, EVB, and EVD by targeting viral 2C proteins from the Prestwick chemical library (Ulferts et al., 2016). Our findings are in line with these previous reports. Another antifungal drug, itraconazole (Hashem et al., 2023), has been identified as an inhibitor of enterovirus replication by targeting the oxysterol-binding protein and viral 3A protein (Gao et al., 2015; Strating et al., 2015). We have also demonstrated the broad inhibitory effect of itraconazole on EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT. Micafungin, an antifungal therapy drug, has been reported to effectively inhibit life-threatening EV-A71 (Kim et al., 2016), and we have confirmed its inhibitory effect on not only EV-A71 but also CVA10 and CVA16, although it's not effective against CVA7. Suramin, an FDA-approved drug used to treat various diseases, including pediatric diseases, exhibits broad-spectrum inhibition of EVAs, including EV-A71 (Ren et al., 2014). However, our study showed that CVA7 infection could not be effectively inhibited by Suramin. Considering the potential influence of the HiBiT insertion on viral sensitivity to drugs, we tested the antiviral activity of micafungin and suramin against wild-type CVA7. We found that these two drugs exhibited similar inhibitory effects on CVA7 infection as they did on CVA7-HiBiT, indicating that the HiBiT insertion did not affect the sensitivity of the recombinant virus to these drugs. The specific mechanisms underlying CVA7 resistance to micafungin and suramin still need to be further elucidated in future research. Suramin can bind to the positively charged region near the 5-fold axis of the EV-A71 capsid, thereby inhibiting the interaction between the virus and cell surface attachment receptors. The amino acid residue at position 145 of VP1 in this region influences the sensitivity of EV-A71 to suramin (Ren et al., 2017). Through comparing the amino acid sequences of CVA10, EV-A71, CVA7, and CVA16, we found that CVA10, CVA7, and CVA16 have a conserved Glu (E) at this position, while EV-A71 has an Arg (R) (Supplementary Fig. S1). While it was expected that CVA7 would be more susceptible to suramin than EV-A71, our data showed the opposite, indicating that the sensitivity of enteroviruses to suramin is influenced by multiple factors. Moreover, the unique sensitivity of CVA7 to micafungin and suramin compared to other enteroviruses emphasizes the difficulty in developing broad-spectrum antiviral drugs for diverse enteroviruses. On a positive note, our EVAs-HiBiT recombinant viruses exhibit strong genetic stability, convenient detection, and similar virological characteristics to wild-type viruses, making them a powerful tool for large-scale antiviral drug screening. Moreover, HiBiT is not limited to drug screening applications. Researchers such as Miyakawa, C. Ilkow, and others have used HiBiT tags for the rapid screening of SARS-CoV-2 antibodies in vitro (Miyakawa et al., 2020; Rezaei et al., 2021), while Laura Mezzanotte's team has applied HiBiT tags for in vivo imaging analysis in mice (Gaspar et al., 2020). These findings also indicate the tremendous potential of the EVAs-HiBiT recombinant virus we have constructed for neutralizing antibody screening and in vivo imaging.

5. Conclusions

In summary, we provide a universal design for construct HiBiT-tagged enteroviruses, which significantly improved the rescue efficiency of recombinants. The HiBiT-tagged enteroviruses were genetically stable and exhibited minimal impact on virus infectivity, replication kinetics, or sensitivity to antiviral drugs. This integration of HiBiT into enteroviruses provides a valuable and convenient tool for advancing research in enterovirus virology.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics statement

This article does not contain any studies with animal or human subjects performed by any of the authors.

Author contributions

Shuye Zhang: conceptualization, supervision, funding acquisition, writing-reviewing and editing, Resources. Jianqing Xu: conceptualization, supervision, Resources; Xiaoyan Zhang: conceptualization, supervision, Project administration. Rui Yu: Roles/Writing -original draft, investigation, methodology. Xiaohong Li: Software, Roles/Writing -original draft, investigation, methodology. Peng Zhang: investigation, methodology, Software. Minghao Xu: Software, investigation. Jitong Zhao: investigation, methodology. Jingjing Yan: methodology, Resources. Chenli Qiu: methodology. Jiayi Shu: methodology. Shuo Zhang: methodology. Miaomiao Kang: methodology.

Conflict of interest

The research was conducted in the absence of any commercial or financial relationships. The authors declare that they have no conflict of interests.

Acknowledgements

The authors would like to Yongkang ​Chen from Fudan University for his kindly help on antiviral drug experiments. This research was supported by the National Natural Science Foundation of China (grant nos. 82002135 and 82172250).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2024.03.004.

Contributor Information

Xiaoyan Zhang, Email: zhangxiaoyan@fudan.edu.cn.

Jianqing Xu, Email: xujianqing@fudan.edu.cn.

Shuye Zhang, Email: shuye_zhang@fudan.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1
mmc1.docx (70.3KB, docx)

figs1.

figs1

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

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