Spodoptera frugiperda (Sf9) cells were cultured in Grace’s medium (Invitrogen, Carlsbad, CA, United States) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) at 27°C; Vero and BHK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY, United States) supplemented with 10% heat-inactivated FBS at 37°C with 5% CO₂. Human rhabdomyosarcoma cells (Rd) and human neuroblastoma cells (Sk) were grown in minimum essential medium (MEM; Life Technologies, Grand Island, NY, United States) containing 10% FBS at 37°C with 5% CO₂. The EV71 BrCr strain was provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences. The polyclonal antibody against EV71 VP1 for Western blotting was prepared by our laboratory, and the monoclonal antibody used for immunohistochemistry was purchased from NZK Biotech (Wuhan, China). β-actin antibody was purchased from Proteintech (Wuhan, China).

The donor plasmid pFB-CMV-Ef1-α was derived from pFastBacTM Dual (Invitrogen, Carlsbad, CA, United States) and replaced the p10 promoter (Pp10) and polyhedrin promoter (PpH) with cytomegalovirus and the Ef1-α promoter (Bai et al., 2008). The infectious cDNA clone pEV71-BrCr-TR (AB204852) used for PCR was kindly supplied by Dr. Arita (Arita et al., 2005). The pEV71-GFP-BrCr cDNA clone, in which a GFP gene was inserted between the 5′ UTR and N-terminus of the VP4 gene of pEV71-BrCr-TR was constructed as previously described (Shang et al., 2013). The T7 RNA polymerase gene was amplified by PCR from BL21 (DE3) using the specific primers T7-F: 5′-ATCGTCGACGCCACCATGAACACGATTAACATCG-3′ and T7-R: 5′-CAAAGCTTTTACGCGAACGCGAAGTCCGAC-3′ (underlined and italicized sequences are SalI and HindIII sites, respectively). After sequence confirmation, this gene was cloned into pFB-CMV-EF1-α using the SalI and HindIII sites and named as pFB-CMV-T7-EF1-α. The cDNA from the 5′ end to the nucleotide position 2,915 with a T7 promoter before the 5′ end of the BrCr genome was amplified using pEV71-BrCr-TR or pEV71-GFP-BrCr as the template, and the primers used were as follows: BrCr-2915-F: 5′-TCAATGCATGCTCGACTGGCTTATCGAAATTACG-3′ and BrCr-2915-R: 5′-TACCCGGGTGGAACAAACAT-3′ (underlined and italicized sequences were NsiI and SmaI sites, respectively). After digestion with NsiI and SmaI, the PCR product was cloned into pFB-CMV-T7-EF1-α. The rest of the cDNA of BrCr obtained from the pEV71-BrCr-TR was cloned into the above construct following the digestion by MIuI and SmaI, resulting in the donor bacmids pFB-T7-BrCr and pFB-T7-GFP-BrCr, which contained the full-length cDNA clone of BrCr. All sequences of the intermediate product were validated by sequencing before use in the subsequent cloning step.

The recombinant viruses (Ac-T7-BrCr, Ac-T7-GFP-BrCr) were generated by transfecting the indicated bacmids into Sf9 cells according to the manufacturer’s instructions (Bac-to-Bac baculovirus expression system, Invitrogen, Carlsbad, CA, United States). The recombinant baculovirus and wild-type virus were collected 60 h after infection, and cell debris was removed by centrifugation at 4,000 × g for 20 min. The supernatant was filtered through a 0.45-mm filter (Millipore, Burlington, VT, United States) and subsequently centrifuged at 25,000 rpm at 4°C for 90 min (BeckmanSW28 rotor) in 5 ml of a 30% (w/v) sucrose cushion in TE buffer (Wang et al., 2010). The virion pellet was resuspended in TE buffer, sucrose was removed by ultrafiltration (100 kDa, Millipore, Burlington, VT, United States), and the pellet was suspended in PBS and stored at –80°C. The viral titer was determined by the 50% tissue culture infectious dose (TCID₅₀) assay using the Reed–Muench method (Svensson et al., 1999).

Mammalian cells were seeded in six-well culture dishes at a concentration of 2 × 10⁵ cells per well. Eight hours after adherence, the medium was removed and replaced with virus (vAc-T7-BrCr, vAc-T7-GFP-BrCr, or wild-type AcMNPV) at a multiplicity of infection (MOI) of 50, and the cells were incubated for 6 h at 37°C. Then, the cells were washed and replaced with 2 ml of fresh medium.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. First-strand cDNA was reverse transcribed from 2 μg of total RNA using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time PCR was conducted with SYBR Green Master Mix (Bio-Rad) on a CFX Connect Real-Time PCR Detection System from (Bio-Rad, Hercules, CA, United States). Specific primers used for qRT-PCR were previously described (Zheng et al., 2013), and GAPDH mRNA was measured as a control.

Cells were harvested and lysed with Western lysis buffer (Beyotime, Shanghai, China). After electrophoresis, proteins were transferred to PVDF membranes (Millipore, Burlington, VT, United States) for 30 min by semidry electrophoresis transfer (Bio-Rad). Then the membranes were blocked in 20 mM Tris–HCl buffer (pH 7.4) containing 37 mM NaCl with 5% BSA (Sigma-Aldrich, St. Louis, MO, United States) at 37°C for 1 h. After that, the film was incubated with polyclonal antibodies against VP1 (diluted 1:1,000 in TBS containing 0.1% Tween 20 and 2% BSA; our lab performed) and β-actin (1:1,000; Proteintech, Wuhan, China) overnight at 4°C. After three washes in TBST for 10 min each time, the membrane was incubated with AP-conjugated secondary antibody (Boster, Wuhan, China) for 2 h at room temperature. Finally, the signals were detected using a BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China).

Thirty-six hours post-transduction with vAc-T7-BrCr, Vero and Rd cells were fixed at 4°C with 2.5% (W/V) glutaraldehyde in 0.1 mol/L PBS (pH 7.2) overnight. Then the electron microscopy samples were prepared as previously described (Wang et al., 2010) and observed by transmission electron microscopy (FEI Tecnai G2, operated at 200 kV).

After transducting Vero cells with v-Ac-EV71, a rescued EV71 was obtained, and Rd cells were infected with the virus to amplify and obtain the F4 generation of virus. Rd cells were seeded in 12-well plates (2 × 10⁵ cells per well, and the confluence reached approximately 60% the next day). The cells were separately infected with wild-type EV71 (WT EV71) or rescued EV71 (rEV71) at an MOI of 1. After 2 h of adsorption and washing with PBS twice, the supernatant was replaced with 2 ml of MEM + 10% FBS. Then the supernatant was collected at 6, 12, 16, 24, and 30 h postinfection, and viral titers were quantified by TCID₅₀ assay.

Wild-type EV71 or rEV71 with the same TCID₅₀ was diluted 1:10 by mixing 80 μl of virus sample with 720 μl of MEM. Two hundred microliters was added to individual wells of 12-well plates containing confluent Rd cells (2 × 10⁵ cells per well, the plates were plated 1 day in advance, and a cell monolayer was formed 24 h later). After infection, the plates were adsorbed for 2 h and washed twice with PBS, and 1.5 ml of 2 × MEM (containing 4% fetal bovine serum) containing 1% agarose was added to individual wells of 12-well plates. Each well was fixed with 1 ml of crystal violet containing formaldehyde and dyed overnight after the plates were incubated at 37°C with 5% CO₂ for 48 h. The numbers and morphology of plaques were recorded after the overlaying layer was flushed away with running water the next day.

One-day-old ICR mice were obtained from the Centers for Disease Prevention and Control of Hubei Province and maintained under specific pathogen-free (SPF) conditions. The mice were randomly divided into the experimental group and the control group (10 mice each). A total of 1 × 10⁷ TCID₅₀ purified recombinant baculoviruses Ac-T7-BrCr and AcMNPV in a volume of 20 μl or PBS was intracerebrally inoculated into mice using a 50-μl Hamilton injector (Hamilton Co., Reno, NV, United States). All experimental procedures were conducted according to the guidance of the Institutional Animal Care and Use Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (No: WIVA07201502).

Brain samples were collected 7 days after inoculation. The samples were washed with sterilized PBS and dissected in half along the coronal line; one half was stored at –80°C, and the other was fixed in 4% paraformaldehyde for immunohistochemistry detection. For the RT-PCR assay, the samples were suspended in TRIzol for RNA extraction. Brain samples suspended in PBS were disrupted by a microelectric tissue homogenizer (Kimble, United States) and sterilized with a 0.22-μm filter for virus isolation in Rd cells.

Brain samples were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) were sliced by a section cutter (Leica, Wetzlar, Germany) and then immunostained overnight with anti-EV71 mAb (1:500) against VP1 at 4°C. Subsequently, the positive cells were visualized with a PV-6002 HRP-conjugated goat anti-mouse detection kit (Zhongshan, Beijing, China) and recorded by a light microscope. The immunofluorescence assay was carried out as previously reported (Pham et al., 2019; Tang et al., 2020). In short, the infected or non-infected Vero cells were washed by PBS three times, followed by fixing with 4% paraformaldehyde for 15 min and permeabilizing for 15 min with 0.5% Triton X-100. Subsequently, samples were incubated with VP1 antibody (1:200) at 4°C overnight. Finally, the positive cells were visualized with Alexa Fluor 647 Conjugate anti-mouse immunoglobulins (Cell Signaling Technology, Danvers, MA, United States) and DAPI (Beyotime, Shanghai, China) and recorded by a Leica confocal microscope.

The construction strategy for the transfer vector of pFB-T7-BrCr and pFB-T7-GFP-BrCr is as shown in Figure 1. Then recombinant bacmid DNA was transfected into Sf9 cells to produce recombinant baculovirus virus, and the virus Ac- T7-BrCr, which can express T7 RNA polymerase in mammalian cells and contains the T7 promoter before the genome of BrCr was constructed using a Bac-to-Bac expression system. Furthermore, to observe whether infectious EV71 can be synthesized after transduction, we constructed the baculovirus Ac-T7-GFP-BrCr carrying a GFP gene in the genome of EV71 as previously described (Shang et al., 2013).

To obtain the recovered infectious virus from the full-length cDNA clone, we transduced Vero cells with the baculoviruses Ac-T7-BrCr, Ac-T7-GFP-BrCr, or AcMNPV. Rescued EV71-induced cytopathic effects (CPEs) and fluorescence appeared 18 h posttransduction, and approximately 80% of the cells were rounded and lost normal cell morphology 48 h posttransduction compared with the controls. This result indicated that EV71 cDNA with a 5′-terminal T7 promoter was efficiently transcribed to generate infectious virus that replicated in permissive Vero cells and showed a specific CPE (Figure 2). At the same time, the culture medium from the transfected cells was collected, and viral titers were determined to be 3.6 × 10⁶ and 1.8 × 10⁶ TCID₅₀/ml for Ac-T7-BrCr and Ac-T7-GFP-BrCr, respectively. To further confirm the recovered EV71, an immunofluorescence assay was performed to analyze the expression of viral structural protein VP1. The immunofluorescence assay results showed that, consistent with the VP1 expression of WT BrCr, the VP1 of recovered viruses was also detected (Supplementary Figure 1).

To confirm the EV71 recovery, we performed Western blotting to examine the expression of VP1, the major structural protein for EV71 in several mammalian cells (Figure 3A). Forty-eight hours posttransduction with Ac-T7-BrCr or AcMNPV at an MOI of 50, the debris of Vero, Rd, Sk, and BHK cells was collected for analysis. The results showed obvious signals in the Vero, Rd, and Sk cells, while an indistinguishable band was observed in the BHK cells. The cells transduced with wild-type AcMNPV were also analyzed as a negative control.

The generation of viral RNA was subsequently confirmed by real-time PCR. Consistent with the Western blotting results, as shown in Figure 3B, viral RNA in Vero cells transduced with Ac-T7-BrCr was clearly detected and was 10 times higher than that in Rd cells, but the other two cell lines showed detectable levels. This result revealed that recombinant baculovirus delivered the genome to the mammalian cells, but the efficiencies in these three EV71 susceptible cells were different.

Observation of viruses by electron microscopy was a direct evidence for rescued viruses. Therefore, Vero and Rd cells were examined using electron microscopy after transduction with baculovirus Ac-T7-BrCr. TEM images showed that large scales of viruses were in the cytoplasm and had a diameter of approximately 30 nm (Figure 3C), which was not observed in the control group that was transduced with wild-type baculovirus ACMNPV (Supplementary Figure 2).

To investigate whether rEV71 could produce infectious viruses, we used one step growth curve and plaque assay. As shown in Figure 4A, rEV71 showed proliferation rates similar to those of WT EV71 when an MOI of 1 was used to infect Rd cells. At 24 h postinoculation, both strains of EV71 reached peak viral titers, which subsequently declined at 30 h postinoculation. Additionally, similar plaque morphology and number were observed between WT EV71 and rEV71 (Figure 4B).

To identify the reconstruction of EV71 in vivo, we intracranially injected the baculoviruses Ac-T7-BrCr and AvMNPV into 1-day-old ICR mice. The transcription and translation of VP1 were detected by RT-PCR and immunohistochemistry methods 7 days after inoculation. A definite fragment was amplified from brain samples by RT-PCR, and the sequence was exactly identical to that of the parental virus (Figure 5A). Additionally, the immunohistochemical expression and localization of VP1 were noted in brown for tissues from the brains injected with Ac-T7-BrCr, while no signal was observed in the control group, which indicated the existence of the rescued virus (Figure 5B). Furthermore, at 36 h postincubation of purified supernatant from mouse brains with Rd cells, an obvious CPE was detected, which further demonstrated the infectivity of the rescued virus (Figure 5C).