African green monkey kidney (Vero) cells used in this study were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in presence of 5% CO₂. The CA16 strain OP293089 was isolated in the Biosafety Laboratory of Shanghai Public Health Clinical Center.

The CA16 viral RNA was extracted with QIAamp Viral RNA Mini Kit (Qiagen, Germany) and reversed transcribed with oligo dT and HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). Using the viral cDNA as the template, three viral fragments: P1, P2, and P3 were amplified. Then, the P1 fragment was cloned in the pcDNA vector (pcDNA-P1), and P2 and P3 fragments were cloned in the pUC57 vector (pUC57-P2+P3). Next, P1 and P2+P3 two fragments were ligated with the pSVA vector to form the infectious DNA clone of CA16 (rCA16). To get the CA16 infectious clone with a Nluc reporter gene (rCA16-Nluc), the Nluc sequence was inserted between 5′UTR and VP4. The genetic stability of Nluc gene was examined by PCR amplification and sanger sequencing, the PCR parameters and primers for Nluc PCR/sequencing were described in the Supplementary Tables 1, 2. The detailed process of the clone construction and the primers used were described in the Supplementary File and Supplementary Table 3.

To generate viral mRNA, the vectors of rCA16 and rCA16-Nluc infectious clone was first linearized by Sal I restrictive enzyme (NEB, The United States), then purified as the template for in vitro transcription using T7 Transcription Kit (Novoprotein, China) according to the manufacturer’s instruction. Next, the purified viral mRNA was transfected to Vero cells by Lipo3000 (Thermo Fisher Scientific, The United States) and incubated for 6 h at 37°C before the medium was changed to fresh complete medium.

Cells for WB detection were harvested and lysed in 1 × SDS loading buffer (NCM Biotech, China) and heated for 10 min at 100°C. Next, cell lysates were loaded and separated by 10% SDS-PAGE gel and transferred to PVDF membrane. Then, the PVDF membrane was blocked with 5% milk, and stained with the viral 2C monoclonal antibody (Youke, China; Liu et al., 2021) and then HRP-Mouse secondary antibody (Yeason, China) to probe viral protein.

Vero cells were seeded on coverslips overnight and infected with the virus at MOI = 1, for another 8 h. Then, the infected cells were washed with cold PBS twice and fixed with 4% paraformaldehyde (Sigma, St Louis, United States) for 15 min. After permeabilization with 0.05% Triton X-100 in 2% FBS, the cells were stained with the 2C or 3D antibody for 1 h. Next, cells were washed with PBS twice and incubated with secondary antibodies AlexoFluo594 for 30 min, followed by washing three times. Finally, cells were stained with DAPI and analyzed using EVOS^® FL Color Imaging Systems (Life technology, The United States).

The virus was 10-fold serially diluted and added to Vero cells in a 12-wells plate, incubated for 2 h at 37°C, then washed with PBS twice, and loaded with 2 × DMEM (4% FBS) and an equal volume of Avicel (IMCD, China). Afterwards, the 12-wells plate was incubated at 37°C for another 3 days. Then, the plate was fixed with 4% paraformaldehyde for 2 h and stained with 1% crystal violet for another 2 h.

For testing virus replication inhibitors: 2.5 × 10⁴ Vero cells were seeded in 96-well plates and incubated overnight, before infection with rCA16-Nluc virus at MOI = 0.1. Two hours post-infection, culture medium was replaced with fresh DMEM medium containing different concentrations of GuHCl or ribavirin for 24 h. Next, luciferase activity was measured according to the manufacturer’s instructions.

For testing virus entry inhibitors: 2.5 × 10⁴ Vero cells were seeded in 96-well plates and incubated overnight, then the culure medium were replaced with DMEM medium containing different concentrations of chloroquine or NH₄Cl for 2 h at 37°C. Afterwards, the cells were infected with rCA16-Nluc virus at MOI = 0.1 for 2 h; then the culture medium was replaced with fresh maintaining DMEM medium for 24 h before measuring the luciferase activity.

After being treated with various concentrations of chemical inhibitors, cell viability was analyzed by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan). Specifically, 3 * 10⁴ Vero cells were seeded in 96-well plates and cultured overnight. Then various concentrations of GuHCl, ribavirin, NH₄Cl, and chloroquine in 2%FBS DMEM were added as triplicates per condition. The next day, the CCK-8 solution was added to each well and incubated at 37°C for 1 h, after which the absorbance at 460 nm was measured (Cytation5, Biotek, The United States).

Twenty-five 7-day-old C57/B6 suckling mice were randomly divided into five groups, including two pCA16 infection groups (n = 5), two rCA16 infection groups (n = 5), and one PBS control group (n = 5). Mice of the infection group were inoculated with 10⁵ TCID₅₀ pCA16 and rCA16 viruses via intraperitoneal routes, while the control mice were given the same volume of PBS. The body weight of mice was followed each day post-infection. For histology and viral characterization, one group of control, pCA16 and rCA16 mice were sacrificed at 4 days after infection. The brain, thigh muscle, lung, and heart tissues were harvested for viral RNA measurement by q-PCR or Hematoxylin and Eosin (HE) staining for tissue damage evaluation.

For generation of neutralizing antibodies, five 6–8 weeks old C57/B6 mice were challenged (i.p) with 10⁵ TCID₅₀ CA16 viruses twice at a 2-week interval, and the control mice were given the same volume of PBS. Serum samples were collected through the ophthalmic vein in the second and fourth weeks after the primary immunization.

To inactivate complement, the collected sera were heated at 56°C for 30 min, and then were 2-fold serially diluted (1:50–1:25,600) in DMEM and incubated with an equal volume of 10³ TCID₅₀ rCA16-Nluc at 37°C for 2 h. Uninfected cells as a negative control, and sera from the PBS group as the positive control. The virus-sera mixtures were added to the 96-wells plate where 2.5 × 10⁴ Vero cells/well were seeded overnight, for a 24 h-incubation at 37°C. Afterwards, the luciferase activities were measured. The nAb titers are derived from the highest dilution index to achieve a 50% reduction of the relative luciferase activity. In addition, all the immunized sera were also confirmed by the CPE-reduction observation.

All the viral RNA copies, luciferase assay data were obtained from at least three repeated experiments. The data were analyzed using Prism 7.0 software (GraphPad, United States) and are presented as the means ± SEM. Statistical significance between the two groups was determined by unpaired two-tailed Student’s t-test. Differences were considered to be not significant (ns), p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

The CA16 strain OP293089, belonging to the B1b subtype (Supplementary Figure 1) was isolated by the Biosafety Laboratory of Shanghai Public Health Clinical Center. To construct the CA16 infectious clone, the viral RNA was extracted, reverse transcribed to single-strand cDNA, and cloned into pSVA vector which contains a T7 promotor as shown in Figure 1A. We generated viral mRNA from the infectious clone by in vitro transcription, and found that the recombinant CA16 viruses (rCA16) could effectively infect Vero cells and cause significant cytopathogenic effect (CPE) at 24 h post-infection (Figure 1B). Western blot using the antibody against viral 2C protein showed the presence of a 35 kDa band, indicating the viral 2C protein was produced (Figure 1C). Besides, an immunofluorescence assay was used to detect the expression of viral 3D protein, showing specifically positive signals in cells infected by rCA16 and pCA16 (Figure 1E). Shown in Figure 1D, rCA16 produced plaques similar to pCA16 in both morphology and numbers. To further characterize the rescued rCA16 viruses, we compared the growth characteristic of rCA16 and pCA16 in Vero cells, both viruses proliferated rapidly after infecting the cells and reaching the plateau level at 24 h post-infection (Figure 1F). Together, these data suggest that we have successfully rescued a rCA16 clone which showed comparable virological characteristics as its parental virus.

Enteroviruses usually infect young children. To examine the virulence and pathogenicity of rCA16, we challenged the one-week-old C57/B6 neonatal mice with 10⁵ TCID₅₀ rCA16 and pCA16, respectively. As shown in Figure 2A, the body weight of mice in the PBS group increased gradually, whereas those in rCA16 and pCA16 groups lost weight from 3 to 4 days after infection and also developed paralysis of the hind limb (Supplementary Figure 2). Severe pathogenicity is indicated by the survival curve, mice infected with rCA16 and pCA16 started dying on day 3 and reached 100% mortality after 5–6 days (Figure 2B). We analyzed the viral loads in various tissues and found that the muscle had the highest viral load, up to 10⁸ viral copies/mg, followed by the brain, heart, and lung tissues (Figure 2C), indication of highly productive viral replication in the infected mice. Severe tissue damages were shown by HE staining (Figure 2D). Infection with rCA16 and pCA16 caused hemorrhagic inflammation of the brain and heart, significant thickening of the alveolar wall, and obvious fragmentation of muscle fibers.

Together, our data showed that this CA16 strain and its recombinant virus are highly pathogenic in one-week-old mice, which provides a convenient pathogenic murine model for enterovirus infection.

Reporter viruses are useful tools to study viral pathogenesis and screen antiviral drugs. We next constructed a CA16 infectious cDNA clone with a Nanoluc luciferase reporter gene (Nluc). Nluc was inserted following 5′UTR and fused with the viral 2A cleavage site (AITTLG) before the VP4 protein (Figure 3A). Then the Nluc-expressing infectious clone was in vitro transcripted into viral mRNA to produce the recombinant CA16 reporter viruses (rCA16-Nluc) by transfection in Vero cells. Vero cells infected with rCA16 and rCA16-Nluc showed significant CPE 24 h after infection (Figure 3B), although the plaques of Nluc-virus are indeed smaller than the rCV16 (Figure 3D), in line with other earlier reports on engineered enteroviruses. Using immunoblotting and immunofluorescence, we detected the expression of viral 2C and 3D proteins at 24 h post-infection in Vero cells (Figures 3C,E). Furthermore, rCA16-Nluc viruses showed similar growth patterns of viral RNAs with rCA16 (Figure 3F). More importantly, besides the viral RNA copies, we also found that luciferase activity increased exponentially following the early stage of infection and reached plateau levels from 12 h post rCA16-Nluc infection, showing a similarly growing kinetics with viral RNAs, whereas rCV16 infection produced only background luciferase signals (Figure 3F). Finally, we also demonstrated that the one-week-old mouse infected with rCA16-Nluc can be monitored through in vivo bioluminescence imaging (Supplementary Figure 3). Together, our data indicate that the insertion of Nluc reporter gene only slightly affects the fitness of rCA16, and that the luciferase activity from rCA16-Nluc closely correlates with its viral replication activities.

The genomic stability of genetically engineered enteroviruses is challenging and unpredictable. To ensure the stability of rCA16-Nluc, the rescued virus was continuingly passaged and monitored in Vero cell cultures, as indicated in Figure 4A. We measured the luciferase activity and quantified Nluc gene copies from passages of P2, P4, P6, P8, and P10 (Figure 4B). Indeed, we found that the gene and activity of the Nluc reporter were maintained at constant levels throughout these extended cultures. We then analyzed the size of the Nluc gene inserts through PCR. As indicated in Figure 4C, the amplified products from P0 to P10 showed the same expected size, indicating no deletion of Nluc gene during the continuous passages. Furthermore, Sanger sequencing of the PCR products verified that the sequence of Nluc gene from P0 to P10 is unchanged (Figure 4D). As a result, we confirmed that the rCA16-Nluc virus is a genetically stable modified enterovirus.

Luciferase has a great advantage over fluorescent protein as the reporter gene in high-throughput screening assays. To see if the rCA16-Nluc can be applied in the settings of high-throughput drug screening, we chose to test two viral replication inhibitors GuHCl and ribavirin (Murray and Nibert, 2007; Li et al., 2008), and two endosomal acidification inhibitors chloroquine and NH₄Cl (Lee et al., 2014; Tan et al., 2018). Indeed, we found that the increased concentrations of the tested inhibitors increasingly inhibited luciferase activity in Vero cells infected with rCA16-Nluc (Figures 5A,C,E,G). Based on this assay, the IC₅₀ of GuHCl, ribavirin, chloroquine, and NH₄Cl were calculated to be 0.07 mM, 0.136 mM, 1.83 μM, and 0.726 mM, respectively, (Figures 5B,D,F,H). Besides, we also measured the CC₅₀ and SI index of each antiviral inhibitor. Together, we have demonstrated that rCA16-Nluc is more cost-effective and faster, and has great application value in high throughput drug screening.

Neutralizing antibodies are widely used to prevent and treat viral infections. The neutralizing antibody titer is classically calculated according to the Reed-Muench algorithm analyzing the formation of CPE, called CPE-Nab assay here. However, this method is not suitable for titration of a large number of samples because it is labor-intensive and cumbersome. Thus, we developed a simple assay (called RLU-Nab assay) to measure neutralizing antibody titers based on the rCA16-Nluc reporter virus. To this end, the anti-CA16 serum was produced by immunizing the C57/B6 mice twice with CA16 as shown in Figure 6A. Then the mouse serums were collected 2 or 4 weeks after the virus challenge, and tested for CA16 neutralizing activity. The neutralizing antibody titers measured in both tests are highly consistent (R² = 0.9699, Figure 6B), suggesting a high quality of the RLU-Nab assay. Our data also showed that the neutralizing antibody titers after the second viral challenge were significantly higher than those of the first challenge (Figure 6C). Thus, the RLU-Nab assay based on rCA16-Nluc is an easy method and has the potential to be used in high-throughput antibody tests.