Cell lines

Human rhabdomyosarcoma (RD, ATCC# CCL-136), HEK293T/17 (ATCC# CRL-11268) and Verda Reno (Vero, ATCC# CCL-81) cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM L-glutamine.

Viruses

The CVA6-TW-00141 strain (GenBank accession no. KR706309, D1 subtype) and the EV71-FJ10 strain were respectively isolated from HFMD clinical specimens in Fujian Province, China. The CVA6-HLJ11 stain (GenBank accession no. MN845762, D2 subtype), CVA6-YN17 strain (GenBank accession no. MN845882, D3 subtype) and CVA6-GD13 strain (GenBank accession no. KF682363, D3 subtype) infectious clones were respectively prepared by transfecting mRNA transcripts, which were synthesized in vitro using a MEGAscript T7 transcription kit (Thermo Fisher Scientific) from linearized full-length cDNA, into RD cells, as previously described [30]. The rescued viruses were propagated in RD cells at 37 ℃, and the supernatants were harvested by freeze-thawing and stored in aliquots at -80 ℃.

Mice

BALB/c mice were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd, China. All mice were maintained in a specific-pathogenfree (SPF) facility of Xiamen University. For infection studies, mice were maintained in animal biosafety level 2 (ASBL-2) facility at 23 ℃. All mice were allowed free access to sterilized water and irradiated diet, and provided with a 12 h light-dark cycle. Animal protocols were performed in accordance with the guidelines of the Xiamen University Institutional Committee for the Care and Use of Laboratory Animals, and approved by the Xiamen University Laboratory Animal Center (approval code: XMULAC20160049).

Construction of a KRM1 expression plasmid

The fragments containing the coding sequences of human KRM1 (GenBank accession no. BC063787) were synthesized from Sangon Biotech. The KRM1 gene, flanked by 5’ Xba I and 3’ Pac I sites, was then cloned into pLV-EF1α-IRES-Puro vector, thus creating a pLV-EF1α-KRM1-IRES-Puro plasmid. The constructed plasmid includes the KRM1 gene with a EF1α promoter, as well as a puromycin gene for the selection of transfected cells.

Construction of a transfected Vero cell line expressing KRM1

Vero cells were pre-seeded at 1 × 105 cells per well into 24-well plates and transfected with pLV-EF1α-KRM1-IRES-Puro using Lipofectamine®3000 transfection reagent (Invitrogen) following manufacturer’s instructions. After 48 h post-transfection, The transient expression of KRM1 Vero cells were then infected with CVA6 or EV71. A Vero cell line with stable expressing of KRM1 was constructed using the lentiviral transduction system. In brief, recombinant lentiviruses were packaged by co-transfecting with pLV-EF1α-KRM1-IRES-Puro, psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) into HEK-293T/17 cells using Lipofectamine®3000 transfection reagent. After 8 h post-transfection, the medium was changed to DMEM containing 10% FBS. The supernatants were then harvested at 48 h after transfection and then filtrated by a 0.45 μm pore size filter. After infection of Vero cells with recombinant lentiviruses and incubation for genome integration, stably transformed cells were selected with puromycin (1 μg/mL) and cloned. Two clones were selected for further study, Vero-KRM1_#11 and Vero-KRM1_#26.

RNA isolation and RT-PCR

Total RNA was isolated from 1 × 106 cells using a cultured cell RNA extraction kit (MolPure® Cell RNA Kit, YEASEN) following the manufacturer’s instructions. The levels of KRM1 mRNA were quantified by real time RT-PCR using a one-step RT-PCR kit (GenMag Bio). RT-PCR reaction was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). Gene-specific primers (forward, 5′-AGCATCCATACAACACTCTGAA-3′; reverse, 5′-CTTCCAGTAGACACCATC.

-CTC-3′; probe, 5′-FAM-AATAGTTGTGCTCACCCAGGCCC-BHQ1-3′) were used for the RT-PCR experiment. RT-PCR was carried out as follows: 15 min at 50 °C and 15 min at 95 °C, followed by 42 cycles of 94 °C for 15 s and 55 °C for 45 s. Each reaction was performed in triplicate. The pLV-EF1α-KRM1-IRES-Puro plasmid was used as a standard sample. A standard curve, generated from serially diluted samples, was used to quantify the number of copies of the gene.

Western blot

1 × 106 RD, Vero, Vero-KRM1_#11, Vero-KRM1_#26 cells were wash in ice-cold PBS and lysed for 10 min in RIPA lysis buffer (BEYOTIME). The cell lysates were centrifuged at 12,000 rpm for 10 min to remove debris. The suspernatant was loaded onto 4–20% Bis-Tris SDS-PAGE gels (SurePAGE™, GenScript) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% skim milk in PBS for 1 h, the membranes were incubated with primary human KRM1-specific polyclonal antibody (1 μg/mL, LS-B10086, Lifespan Biosciences) for 1 h. After three washes with PBST (0.05% Tween-20 in PBS), the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (GAR-HRP) second antibodies (1:5000 dilution) for 30 min. GAPDH (HRP-conjugated GAPDH antibody, Proteintech, 1:10,000 dilution) was used as the internal reference. After three washes with PBST, the membranes were visualized by chemiluminescence (SuperSignalTM West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific), and the images were obtained by FUSION FX EDGE SPECTRA (Vilber Bio Imaging). All procedures were conducted at room temperature.

Immunofluorescence assay

RD, Vero and Vero-KRM1_#11 cells were pre-seeded at 1 × 105 cells per well into 24-well plates on the 15 mm cover glasses (Nest) and then infected with CVA6 or EV71 at a multiplicity of infection (MOI) of 1. At 12 h post-infection (hpi), cells were fixed with 4% paraformaldehyde for 30 min in the dark and permeabilized with 0.3% Triton X-100 diluted in PBS for 15 min. After blocking with 2% bovine serum albumin (BSA) diluted in PBS at 37℃ for 20 min, cells were incubated with CVA6-specific mAb 4D6 or EV71-specific mAb CT11F9 [31] (4 μg/mL) at 37℃ for 1 h. After three washes with PBST, cells were incubated with Alexa Fluor® 488 goat anti-mouse (GAM-AF488) second antibody (1:1000 dilution) at 37℃ for 30 min in the dark. After three washes with PBST, cells nuclei were stained with 40, 6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, cells were observed and image data were captured using an EVOS M7000 imaging system (Thermo Fisher Scientific).

Viral growth kinetics

RD, Vero or Vero-KRM1_#11 were pre-seeded at 1 × 105 cells per well into 24-well plates and then infected with CVA6-TW-00141 strain at a multiplicity of infection (MOI) of 0.01. Virus propagation was performed at 37℃. At 12, 24, 36, 48, 60, 72, 84, 96 and 108 hpi, one plate was harversted per cell line. Virus was released from the cells by freeze-thawing. After centrifugation, the virus samples were collected and stored at -80℃. Once the final time point was acquired, all samples were titrated using the virus titration assay.

Virus titration assay

Virus titers were determined by the median end-point titration using RD, Vero or Vero-KRM1 cells and expressed as the 50% tissue culture infectious dose (TCID50). In brief, cells were pre-seeded at 5 × 103 cells per well into 96-well plates and then infected with ten-fold serial dilutions of the virus. Each dilution was inoculated with 8 wells for 100 μL per well. After an incubation period of 7 days at 37℃, cells were observed for CPE. The TCID50 values were were calculated according to the Behrens-Kärber method.

Virus production and purification

The virus production and purification experiments were conducted as previously described [32]. In brief, CVA6-TW-00141 strain was grown in RD or Vero-KRM1 cells at a MOI of 0.1 at 37℃, respectively. Virus was harvested 3 days post-infection (dpi), and then centrifuged at 25,000 × g for 30 min to remove the cell debris, and precipitated by using 8% polyethylene glycol (PEG) 8,000 and 0.3 M NaCl in 0.1 M phosphate buffer (PB, pH7.4) at 4℃ for 12 h. After centrifugation, virus was resuspended and then sedimented through a linear 15–45% (w/v) sucrose density gradient at 153,900 g for 4.5 h at 4℃ using a Beckman SW41 Ti rotor. The fractions were collected and independently dialysed against PBS and concentrated by an Ultra-4 centrifugal concentrator (50 kDa, Millipore). The quantity and quality of CVA6 particles were examined by negative-staining electron microscopy (TEM). The protein composition was analyzed by SDS-PAGE.

Vaccine preparation and immunization of mice

The purified CVA6 particles were inactivated by adding β-propiolactone and mixed with an equal volume of aluminum adjuvant. Female BALB/c mice aged 6–8 weeks old (n = 5 per group) were immunized intraperitoneally (i.p.) at a dosage of 1.5 μg of inactivated CVA6 vaccine in a volume of 500 μL. Each mice was immunized at weeks 0 and 2, and subsequently bled at weeks 0, 4, 6, 8 and 10. The blood samples were centrifuged at 12,000 rpm at 4 ℃ for 15 min. The sera were heat-inactivated at 56 ℃ for 30 min, and then stored at -20 °C for ELISA and neutralization assays.

ELISA

The 96-well ELISA plates were coated with 50 ng per well of purified CVA6 particles in PBS. After incubation at 4℃ overnight, the plates were washed once with PBST and saturated with the saturation buffer (0.25% casein and 1% gelatin in PBS buffer) at 37℃ for 2 h. Serum samples were diluted in PBS and added to the plates, followed by incubation at 37℃ for 1 h. After five washes with PBST, the plates were added with GAM-HRP (1:5000 dilution) and incubated at 37℃ for 30 min. For color development, after five washes with PBST, the plates were incubated with a solution of 3,3′,5,5′-tetramethylbenzidine (TMB) at 37℃ for 15 min, and the reaction was terminated with 2 M H2SO4. Finally, the absorbance was measured at 450/620 nm with a plate reader.

In vitro neutralization assay

RD cells were pre-seeded at 1 × 104 cells per well into 96-well plates. Serum samples from mice were two-fold serially diluted ranging from 1:32 to 1:2,048 and mixed with equal volume of CVA6-TW-00141 strain (100 TCID50) in 96-well plates at 37℃ for 2 h. Virus-serum mixtures were added into the cell well and then incubated at 35℃ for 7 days. Each well was observed by microscope. The neutralization titers were the averages of the triplicate readings calculated based on the highest dilution in over 50% cytopathic effect (CPE). The neutralizing titer of sera with values ≥ 32 was considered the threshold for positivity.

In vivo protection test

One-day-old BALB/c mice (n = 6 per group) were firstly administered i.p. with 100 μL diluted sera 6 h before challenged i.p. with 100 μL of CVA6-TW-00141 strain (5 × 105 TCID50 per mouse). The mice in the control group were treated with PBS via the same route. All mice were monitored daily for survival, clinical illness and weight until 20 dpi. The grade of clinical disease was scored as follows: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limb weakness; 4, hind-limb paralysis; and 5, morbidity and death.

Statistics

Statistical analysis was performed using GraphPad Prism version 8.0. Anti-CVA6 IgG titers and neutralizing titers were presented as the geometric mean titer (GMT) and converted to the logarithmic scale. The results are expressed in terms of the means ± standard deviations (SD) and analyzed by using the unpaired Student’s t-test. The health scores were shown as means. The survival curves were evaluated by the Log-rank (Mantel-Cox) test. A P value < 0.05 was considered statistically significant. Statistical details of the experiments can be found in the Results and Figure legends.