Amphotericin B

We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.com). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 10⁵ cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO₂ and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols (9 (link),10 (link)).
When CPEs were observed, we scraped cell monolayers with the back of a pipette tip. We used 50 μL of viral lysate for total nucleic acid extraction for confirmatory testing and sequencing. We also used 50 μL of virus lysate to inoculate a well of a 90% confluent 24-well plate.

Antibiotic treatment started with three days of amphotericin-B (Bristol Meyers Squibb, New York City, NY) 0.1 mg/ml, administered by gavage 12h⁻¹ (Figure 2A). From day three, water flasks were supplemented with ampicillin (Bristol Meyers Squibb, New York City, NY) 1 g/l and antibiotic concoction consisting of vancomycin (Abbot, Abbot Park, IL) 5 mg/ml, neomycin (Invitrogen, Carlsbad, CA) 10 mg/ml, metronidazol (Actavis, Hafnarfjordur, Iceland) 10 mg/ml, and amphotericin-B (Bristol Meyers Squibb) 0.1 mg/ml was administered by antibiotic gavage 12h⁻¹. Gavage volume of 10 ml/kg body weight was delivered with a stainless steel tube without prior sedation of the mice. Fresh antibiotic concoction was mixed every day and ampicillin and water was renewed every 7th day.

This study was conducted in Damietta Governorate on the Egyptian Mediterranean coast (northern east Nile Delta), Egypt through the period from October 2021 to March 2022. A total of 200 cloacal swabs were collected from migratory and broiler chicken birds. Broiler chickens were selected from poultry farms and live bird markets near which the migratory birds were hunted at the similar time points. One hundred samples were obtained from migratory birds and 100 from broiler chickens; 50 from 5 poultry farms (10 for each farm) with deep litter system and 50 from 3 live bird markets located in different regions inside Damietta Governorate. Five broiler poultry farms were chosen on the basis of their owners’ willingness to permit the samples collection. Broiler chicken birds from the farms and live bird markets were selected randomly. The map of Damietta Governorate was constructed to highlight the location of the selected broiler chicken farms and live bird markets in relation to the rest of Damietta (Supplementary Fig. 9). The migratory birds that were found near to the examined farms and live bird markets were trapped by net traps, sampled, marked (to ensure that each bird was only sampled once) and photographed to detect its species. The cotton swabs were aseptically collected on 2 ml of Bolton broth (Oxoid, UK) then labeled and transported within 1 h in an ice box at 4 °C to the Reference Laboratory for Veterinary Quality control on Poultry production to perform further examinations. All samples were incubated at 42 °C for 48 h under microaerophilic conditions. Isolation and identification of Campylobacter spp.
Each enriched sample was streaked onto modified charcoal cefoperazone deoxycholate agar (Oxoid, UK) with antibiotic solution (cefoperazone sodium salt; 0.032 g, amphotericin B; 0.01 g and water; 5 ml) and incubated at 42 °C for 48 h. The suspected colonies were identified by morphological characteristics and Gram staining [45 ]. The suspected isolates were subjected to standard biochemical procedures, including tests for hippurate, acetate hydrolysis and catalase [46 ].

HeLa cell lines (ATCC CCL2) used were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; 10270106; Thermo Fisher Scientific), 1% Penicillin/Streptomycin (15140122; Thermo Fisher Scientific), and 0.1% Amphotericin B (Fungizone; 11510496; Thermo Fisher Scientific). Cell lines were cultured as a monolayer at 37°C and 5% CO₂. HeLa H2B-GFP, mCherry-Tubulin cell line was generated by transfecting mCherry-Tubulin expressing eukaryotic plasmid vector into HeLa H2B-GFP cells (Draviam et al., 2006 (link)). The HeLa mKate2-EB3 cell line was generated by transfecting a pmKate2-EB3 plasmid vector (#FP316; Evrogen) into HeLa cells. Plasmid transfection was carried out using DharmaFECT duo (T-2010; Dharmacon). The HeLa H2B-GFP, SiR-Tubulin cell line was generated by adding SiR-Tubulin dye, a paclitaxel-based fluorescent compound (Lukinavičius et al., 2014 (link); Spirochrome SC002; 100 nM) just 1 h before imaging. The HeLa FRT/TO cell line expressing siRNA-resistant MARK2-YFP-WT or KD mutant was generated by transfecting a Tet-inducible expression vector encoding siRNA-resistant MARK2-YFP-WT or KD, followed by colony picking (Zulkipli et al., 2018 (link)). Vectors bearing point mutants of MARK2 were generated by polymerase chain reaction–based point mutagenesis and confirmed by DNA sequencing (Hart et al., 2019 (link)). Fluorescent cells were enriched using a BD FACSAria III Cell Sorter for fluorescence-activated cell sorting (FACS).