Antibodies

Mouse anti-chicken Villin monoclonal antibody (sc-58897) and mouse anti-human CGA monoclonal antibody (sc-393941) were purchased from Santa Cruz (Texas, USA). Rabbit anti-human Sox9 monoclonal antibody (82630) and mouse anti-PCNA monoclonal antibody (2586S) were purchased from Cell Signaling Technology (Massachusetts, USA). Rabbit anti-human Muc2 monoclonal antibody was purchased from Abcam (ab134119, Cambridge, UK). Rabbit anti-human LYZ polyclonal antibody was purchased from Invitrogen (PA5-16668, California, USA). Mouse anti-human Ki67 monoclonal antibody was purchased from BD Biosciences (550609, New Jersey, USA). Rabbit anti-GAPDH polyclonal antibody was purchased from Proteintech (10494–1-AP, Chicago, USA). TGEV N was kindly gifted by Prof. Li Feng from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. PEDV N was made in our laboratory. All antibodies were diluted, with a 100-fold dilution applied for IFA and a 1000-fold dilution used for western blot analysis.

Viruses and animals

The TGEV H165 strain was previously recovered from a commercialized attenuated live vaccine and stored in our laboratory (170041133, Keqian Biology, Wuhan, China). TGEV-Miller strain was kindly gifted by Prof. Zhenhui Song from the Department of Veterinary Medicine at Southwest University in Chongqing, People’s Republic of China. The cell-adapted PEDV-LJX strain was obtained from diarrheal piglets from a pig farm in Liujiaxia, Gansu province. Briefly, to isolate the virus, the intestinal contents from diarrheal piglets were collected and resuspended in PBS. After centrifugation, the supernatant was collected and filtered through a 0.45-µm filter. We employed TaqMan probe-based reverse transcription‒quantitative PCR (RT‒qPCR) to screen for several enteric viruses following a previously established protocol in our laboratory [35]. The results indicated a high number of PEDV copies, whereas no other enteric viruses were detected. The supernatant was preserved in -80 °C until further use. For the isolation of crypts and collection of intestinal mucus, porcine ileum samples were obtained from the Luoniushan slaughterhouse in Hainan Province. The pigs were 6-month-old fattening boars of the Large White breed and were fed with commercial feed. All experimental procedures and animal care were conducted in accordance with the approved guidelines for the Care and Use of Laboratory Animals of Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences, China.

Cell lines culture

ST cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; D6429, Sigma, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS; A6901, Invigentech, Nova Lima, Brazil), which are commonly used cell lines for TGEV infection. Vero‒E6 cells were also cultured in DMEM with 10% FBS, which are widely recognized as ideal cell lines for investigating PEDV infection.

Establishment of mature porcine intestinal organoid monolayer

The generation of porcine ileum organoids and development of organoid monolayer was performed following previous protocols [23, 36]. In brief, 0.33cm2 transwell inserts (3413, Corning, NY, USA) were incubated with 0.1% Matrigel (356,231, Corning, NY, USA) in DMEM/F12 (D8437, Sigma, St. Louis, USA) for 1 h at room temperature. After aspiration of the liquid, the transwell inserts were air‒dried for 15 min. Organoids cultured for 5 days were collected and digested into single cells, after which 150,000 cells were seeded into the precoated transwell. Undifferentiated monolayer was maintained with 200 µL of IntestiCult™ Organoid Growth Medium for humans (OGM; 06010, Stem Cell, Vancouver, Canada) supplemented with 10 µM ATP-competitive inhibitor of Rho-associated kinases (Y-27632; 72,302, CST, MA, USA) in the apical chamber and 500 µL of the same medium in basolateral chamber, and cultured for a duration of 3 days. In addition, for differentiated monolayer, OGM supplemented with Y-27632 was required continuously for 7 days, fresh medium was replaced every two days, and then the medium was switched to IntestiCult™ Organoid Differentiation Medium for human (ODM; 100–0214, Stem Cell, Vancouver, Canada). In submerged monolayer, cells were cultured with ODM containing Y-27632 in both apical and basal chambers. In the ALI monolayer, the medium was thoroughly aspirated from the apical chamber, and ODM containing Y-27632 was added only to the basal chamber. Submerged monolayer and ALI monolayer were cultured for 3 days before the next experiment. For the mucus monolayer, the ALI monolayer generated in the above stage was continued to culture for 12 days, and the medium was replaced every 2 days.

Virus infection

The submerged monolayer and ALI monolayer were washed three times with phosphate-buffered saline (PBS) and subsequently infected with the TGEV H165 strain (MOI = 1) or the PEDV-LJX strain (MOI = 1). Briefly, 68 μL of TGEV of the H165 strain (106.5 TCID50/mL) and 12 μL of the PEDV-LJX strain (107.25 TCID50/mL) supplemented with 38 μL of DMEM/F12, were added to the apical surface of the transwell for 1 h. The ST or Vero‒E6 cell culture supernatants were used to treat the mock-infected organoid monolayers, respectively. The unattached viruses were removed by washing with PBS. The infected organoid monolayers were then cultured with ODM containing 10 mM Y-27632, either under submerged conditions or ALI conditions.

Pretreatment with virus prior to infection

The TGEV H165 strain (MO I = 0.1) or PEDV-LJX strain (MOI = 0.1) was preincubated with native porcine small intestinal mucus (22.21 µg/µL, 10 µL), gastric mucin (M2378, Sigma, St. Louis, USA), ALI mucus (8.22 µg/µL, 10 µL), porcine recombinant Muc2 (RPA705Po01, Cloud-Clone Corp, Wuhan, China) or porcine native Muc2 (NPA705Po01, Cloud-Clone Corp, Wuhan, China) for 1 h at 37 °C respectively. ST or Vero‒E6 cells were incubated with the mixture of virus and mucus/Muc2 for 1 h, and then the unattached virus was removed with PBS. The infected cells or supernatants were collected at 16 h post-infection (hpi).

Neuraminidase treatment

The native Muc2 was pretreated with 100 mU neuraminidase (11080725001, Roche, Basel, Switzerland) for 1 h at 37 °C to cleave sialic acid glycans following previous reports [14, 18]. Then the mixture was incubated with the TGEV H165 strain (MOI = 0.1) or the PEDV-LJX strain (MOI = 0.1) for 1 h at 37 °C prior to infection, and a monolayer of ST cells or Vero‒E6 cells was infected with the virus‒mixture for 1 h at 37 °C, respectively. The infected cells were harvested for detection at 16 hpi.

Virus titration

The infectious virus particles were titrated on ST cells or Vero‒E6 cells via the median tissue culture infectious dose (TCID50) assay. The cell supernatants were collected at 16 hpi, diluted with tenfold series dilutions, and added to the cells in 96-well plates. Dilutions and wells containing more than 50% cytopathic cells were recorded, and then the virus titration was counted by the Reed-Muench method [37].

RNA extraction and RT-qPCR

The total RNAs from organoid monolayer, Vero-E6 cells, and ST cells were separately extracted using RNAiso reagent (9109, TaKaRa, Kusatsu, Japan). The concentration of the RNA samples was measured using a NanoPhotometer (Implen, Munich, Germany). Subsequently, cDNAs were synthesized using the Honor II 1st Strand cDNA Synthesis SuperMix (R223-01, Vazyme, Nanjing, China) according to the manufacturer’s instructions. For viral load detection, the TaqMan probe-based RT-qPCR was employed following the protocol previously established in our laboratory [35]. Relative gene expression levels were performed with 2 × SYBR green master mix (Q311-02, Vazyme, Nanjing, China) according to the manufacturer’s instructions. The qPCR parameters were 95 °C for 30 s for initial denaturation; 40 cycles of 95 °C for 10 s, 60 °C for 30 s, followed by a dissociation curve segment (95 °C, 15 s; 60 °C, 60 s; 95 °C, 15 s). Target gene expression was normalized to GAPDH expression, a commonly used housekeeping gene. The relative fold change of the target gene was calculated with the 2−ΔΔCT method. The primers and probes used in this study are listed in Table 2.

Table 2 Primers for real-time qPCRWestern blot

The cells were lysed and harvested using NP40 lysis buffer (P0013F, Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluorid (PMSF; ST505, Beyotime, Shanghai, China) for 20 min at 4 °C after being washed with PBS. The protein samples (20 µL) were loaded and separated on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane (10600023, GE, Boston, USA). The membranes were then blocked with 5% skim milk (232100, BD, New Jersey, USA) for 2 h at room temperature to prevent nonspecific binding. Subsequently, the membranes were incubated overnight at 4 °C with specific primary antibodies, which were diluted in 0.05% PBST (1:1000). After primary antibody incubation, the membranes were washed with 0.05% PBST and then incubated with HRP-conjugated anti-rabbit secondary antibody (ZB-2306, ZSBIO, Beijing, China) or HRP-conjugated anti-mouse secondary antibody (AP160P, Sigma, St. Louis, USA) for 1 h at room temperature, which were diluted in 0.05% PBST (1:5000). Finally, the protein expression was visualized with chemiluminescence detection reagents (K-12045-D50, Advansta, CA, USA).

Immunofluorescence staining

The cells were fixed with 4% paraformaldehyde (P0099, Beyotime, China) for 20 min at room temperature, and the excess fixative was removed by washing with PBS for 3 times. Then the cells were permeabilized with 0.2% Triton X-100 (P0096, Beyotime, Shanghai, China) for 20 min prior to being blocked with 5% BSA (4240, Biofroxx, Frankfurt, Germany) for 1 h at room temperature. Primary antibodies were diluted with PBS (1:100) and incubated at 4 °C overnight. Subsequently, the cells were labeled with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (ab150077, Abcam, Cambridge, UK) or Alexa Fluor 647-conjugated anti-mouse secondary antibody (ab150115, Abcam, Cambridge, UK) for 1 h at room temperature, which were diluted with PBS (1:1000). Finally, the nuclei were stained with with 4′,6-diamidino-2-phenylindole (DAPI; C1002, Beyotime, Shanghai, China). In addition, transwell membranes cut with surgical blades and cell slides were placed on glass slides for imaging. Specific markers were observed via a confocal microscopy (Zeiss LSM 900, Germany).

Periodic acid-Schiff and alcian blue staining

The transwell monolayer was rinsed with PBS and fixed with 4% paraformaldehyde, and then incubated at 4 °C overnight. The filter membrane was cut with a surgical blade after being washed 3 times with PBS, embedded in 1% low-melting agarose solution (ST107, Beyotime, Shanghai, China), and paraffin-embed according to the standard protocol. Periodic acid-Schiff (PAS; C0142S, Beyotime, Shanghai, China) and alcian blue (AB; C0153S, Beyotime, Shanghai, China) staining were performed following the manufacturer’s recommended protocols.

Scanning electron microscopy

Transwells were washed with PBS, fixed overnight at 4 °C with 2.5% glutaraldehyde (BL911A, Biosharp, Hefei, China), and then washed with PBS 3 times. The filter membrane was cut with a surgical blade, dehydrated through a gradient ethanol, and finally, replaced with tert-butanol (25725–11-5, Macklin, Shanghai, China). Samples were then dried for 2 h using a critical point drier and sputter-coated with gold for 60 s. Visualization was performed with a Zeiss Sigma FE-SEM microscopy (Carl Zeiss, Germany).

Collection and treatment of mucus

Hydrated gel-like mucus derived from the ALI monolayer was aspirated with 10 µL tips and stored at − 80 °C. Following the previous report, native small intestinal mucus was scraped from the ileum of the pigs, resuspended in PBS (1:3 dilutions) supplemented with 2% penicillin/streptomycin (P4333, Sigma, St. Louis, USA) and 100 mg/mL gentamicin (G3632, Sigma, St. Louis, USA). The mucus mixture was vortexed on ice for 15 min and centrifuged at 12,000 rpm at 4 °C for 20 min [14]. Subsequently, the supernatant was collected and stored at − 80 °C until further use.

Mass spectrometry analysis and protein identification

The mucus derived from the ALI monolayer and native small intestinal mucus isolated from porcine ileum were collected. Mucus samples were resuspended in SDT (4% SDS, 100 mM Tris–HCl, 1 mM DTT, pH 7.6) buffer for protein lysis and extraction. Protein digestion was performed according to filter-aided sample preparation (FASP) procedure by trypsin. Briefly, 40 mM DTT was added to samples and mixed for 45 min at 37 °C at 600 rpm and alkylation with 20 mM iodoacetamide (IAA) for 30 min in darkness. Subsequently, trypsin was added to the samples (the trypsin: protein (wt/wt) ratio was 1:50) and samples were incubated for 15–18 h at 37 °C. Afterwards, the peptides were desalted on C18 cartridges, concentrated by vacuum centrifugation and dissolved in 0.1% (v/v) formic acid. Finally, LC–MS/MS analysis was performed on a timsTOF Pro mass spectrometry (Bruker, Germany) that was coupled to Nanoelute (Bruker, Germany). The MS raw data for each sample were identified and quantified using the MaxQuant 1.6.14 software. Database searches were performed against the Sus Scrofa UniProt protein database with the following parameters: (1) 2 max missed cleavages; (2) fixed modifications: carbamidomethyl (C); (3) variable modifications: oxidation (M). The false discovery rate (FDR) was set to 0.01 for proteins and peptides. For quantification, label-free quantification (LFQ) and intensity-based absolute quantification (iBAQ) were enabled.

Statistical analysis

The data from three independent biological replicates were presented as the means ± standard deviations (SDs). The statistical analysis was performed by one-way analysis of variance (ANOVA) or Student’s t-test with GraphPad Prism 7 software and are shown as P values.