Formaldehyde

Polyethylenimine (25000 Da) was purchased from BASF, phosphorous acid, formaldehyde, hydrochloric acid and sodium hydroxide were obtained from VWR and dyes (methylene blue, MB, and methyl orange, MO) from Sigma-Aldrich. Dialysis tubings were bought from Roth. Absorbance was measured by using a UV–visible Perkin-Elmer Analyst 100 spectrophotometer. The microwave used was a Prolabo Synthewave. A Varian SpectrAA 55 AAS was used to detect potential traces of iron in the supernatants after collecting nanoparticles. Zeta potential measurements were determined by DLS analysis using a Malvern Zetasizer nanoZS model.

Bovine Serum Albumin (Sigma-Aldrich, USA), Fresh hen’s egg albumin, Complete Freund’s adjuvant (Sigma-Aldrich, USA), Formaldehyde (VWR, International Ltd), Turpentine oil (UNI-CHEM), Aspirin (UNI-CHEM), Diclofenac Sodium (Sigma-Aldrich, USA), Ascorbic acid (MERCK, Darmstadt, Germany). All the other chemicals used were of analytical grade.

Formaldehyde (36%) was purchased
from VWR; phosphorous acid (99%) and hexylamine (98%) from Fluka Chemical
Co.; 2-ethylhexylamine (98%), La(NO₃)₃·6H₂O, and propylamine hydrochloride from Sigma-Aldrich; ethylamine
hydrochloride (98%), butylamine (99%), and Y(NO₃)₃·6H₂O (99.8%) from Merck; and Lu(NO₃)₃·H₂O from abcr and amylamine (98%) from TCI
chemicals. All of the chemicals were reagent grade and used without
further purification. NMR measurements and titrations were performed
on a Bruker Avance III 300 MHz-spectrometer, and NMR data was processed
with Bruker TopSpin 4.0.8. IR spectra were measured by Bruker Alpha
FT-IR. Elemental analyses were done by an Elementar Vario EL III-analysator.
Lanthanoid concentrations were determined by a Perkin Elmer Optima
8300 DV ICP-OES- spectrometer.

Weight was measured daily between 8:00 a.m. and 11:00 a.m. Doxo-treated animals experiencing a weight loss <5% were excluded from the study due to presumed injection error (28 (link), 29 (link)). The mice were anesthetized by IP injection of ketamine 100 mg/kg (Ketaminol® Vet, MSD Animal Health, Boxmeer, The Netherlands) and xylazine 10 mg/kg (Rompun® vet, Bayer Animal Health GmbH, Leverkusen, Germany) and subsequently exsanguinated by collecting a blood sample by cardiac puncture. Animals were perfused with phosphate-buffered saline and a tail biopsy was collected. The small intestine was removed from the pyloric sphincter to the ileocecal junction and divided into three equally sized pieces representing duodenum, jejunum and ileum for use in histopathological analysis, quantitative real time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The length of these segments was measured, and the segments were flushed with phosphate-buffered saline. Samples for histopathological analysis were fixated in 4% formaldehyde (VWR Chemicals, Leuven, Belgium) for 48 h, transferred to phosphate-buffered saline with 0.05% NaN₃, embedded in paraffin, mounted on glass slides, and stained with haematoxylin and eosin (HE) at the Department of Pathology, Odense University Hospital, Odense, Denmark.

VNT on pig sera was done as described previously^{36 (link)}. Briefly, Vero E6 cells were seeded in 96-well flat-bottom plates (1 × 10⁵ cells/mL) and incubated at 37 °C overnight prior to the assays. Two-fold serial dilutions of sera (starting dilution 1 in 5) in quadruplicate were prepared in 96-well round-bottom plates using Dulbecco’s modified Eagle medium (DMEM) with 1% (v/v) FBS and 1% antibiotic–antimycotic (Gibco). 75 µL of the diluted sera was mixed with an equal volume of media containing 64 PFU of SARS-CoV-2 virus (hCoV-19/England/02/2020, EPI_ISL407073) and incubated for 1 h at 37 °C. Media in the wells seeded with Vero E6 was replaced with 100 µL DMEM with 10% (v/v) FBS and 1% antibiotic–antimycotic (Gibco) and 100 µL of the sera–virus mixture was added into the wells. The plates were incubated for 6 days at 37 °C. Cytopathic effect (CPE) was monitored on a brightfield microscopy, and by fixation using formaldehyde (VWR) and staining using 0.1% (w/v) Toluidine Blue (Sigma-Aldrich). CPE was scored by researchers who were blinded to the identity of the samples. No sera or no virus controls were run in parallel on each plate. Neutralisation titres (ND5₀) were expressed as the reciprocal of the serum dilution that prevented CPE in 50% of the wells.

WT or chimeric DENVs in the supernatants of cell cultures were analyzed for their viral copy numbers. Viral RNAs were extracted, using Quick-RNA^TM-Viral kit (Zymo Research). RT reaction was performed to make the viral cDNAs by ProtoScript II reverse transcriptase (NEB) with random primer and dNTPs (NEB) at 42°C for 2 hours. The copy numbers of cDNAs were measured by qPCR with primer pair amplifying capsid or NS1 region (Table S1) and iTaq Universal SYBR green Supermix (Bio-Rad) in Mic qPCR cycler (Bio Molecular System). The average threshold cycle (Ct) values were converted to viral copy numbers by comparing them with the standard amounts of DENV cDNAs.
Plaque assay was performed in paired wells of six well plates, using LLC/MK2 cells, which were infected for 2 hours by WT or chimeric DENV from serially diluted supernatants in the infected C6/36 cell cultures. After removal from medium, cells were overlaid with DMEM containing 0.9% SeaPlaque agarose (Lonza) and were continuously incubated for 9–15 days at 37°C. Plaques were fixed with 37% formaldehyde (VWR) and were visualized by crystal violet staining.

Vero E6 cells were grown in DMEM containing sodium pyruvate and l-glutamine (Sigma-Aldrich, Poole, UK), 10% FBS (Gibco, Thermo Fisher, Loughborough, UK), 0.2% penicillin/streptomycin (10,000 U/mL; Gibco) (maintenance media) at 37 °C and 5% CO₂. SARS-CoV-2 isolate England-2 stocks were grown in Vero E6 cells using a multiplicity of infection (MOI) of 0.0001 for 3 days at 37 °C in propagation media (maintenance media containing 2% FBS). SARS-CoV-2 stocks were titrated on Vero E6 cells using MEM (Gibco), 2% FCS (Labtech, Heathfield, UK), 0.8% Avicel (FMC BioPolymer, Girvan, UK) as overlay. Plaque assays were fixed using formaldehyde (VWR, Leighton Buzzard, UK) and stained using 0.1% Toluidine Blue (Sigma-Aldrich). All work with live SARS-CoV-2 virus was performed in ACDP HG3 laboratories by trained personnel. The propagation, purification and assessment of ChAdOx1 nCoV-19 titres were as described previously⁷ .